U.S. patent application number 14/408799 was filed with the patent office on 2015-06-25 for rapid specific pathogen free animal.
This patent application is currently assigned to BIOVALENCE SDN. BHD.. The applicant listed for this patent is BIOVALENCE SDN. BHD.. Invention is credited to Ag., Muhammad Sagaf Abu Bakar, Eng Huan Ung.
Application Number | 20150173333 14/408799 |
Document ID | / |
Family ID | 49783571 |
Filed Date | 2015-06-25 |
United States Patent
Application |
20150173333 |
Kind Code |
A1 |
Ung; Eng Huan ; et
al. |
June 25, 2015 |
RAPID SPECIFIC PATHOGEN FREE ANIMAL
Abstract
A method of producing at least one specific pathogen free (SPF)
non-human animal and/or a method of producing at least one specific
pathogen resistant (SPR) non-human animal, the method comprising
administration of a fusion protein to the surviving animal wherein
the fusion protein comprises at least one polypeptide B which is a
Type 1 Ribosome Inactivating Protein (RIP) or fragment thereof; and
(i) at least one polypeptide A which is an Antimicrobial peptide;
and/or (ii) at least one polypeptide C which is a Cationic
Antimicrobial Peptide (CAP) or fragment thereof.
Inventors: |
Ung; Eng Huan; (Sabah,
MY) ; Abu Bakar; Ag., Muhammad Sagaf; (Sabah,
MY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOVALENCE SDN. BHD. |
Petaling Jaya |
|
MY |
|
|
Assignee: |
BIOVALENCE SDN. BHD.
Petaling Jaya
MY
|
Family ID: |
49783571 |
Appl. No.: |
14/408799 |
Filed: |
June 25, 2013 |
PCT Filed: |
June 25, 2013 |
PCT NO: |
PCT/MY2013/000116 |
371 Date: |
December 17, 2014 |
Current U.S.
Class: |
800/13 ; 800/21;
800/22 |
Current CPC
Class: |
A01K 2227/105 20130101;
C12N 15/62 20130101; A01K 67/0338 20130101; C12N 15/8509 20130101;
A01K 67/027 20130101; C12N 9/2497 20130101; C07K 14/4723 20130101;
C07K 2319/00 20130101 |
International
Class: |
A01K 67/033 20060101
A01K067/033; C12N 15/62 20060101 C12N015/62; C12N 15/85 20060101
C12N015/85 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 26, 2012 |
MY |
PI 2012002924 |
Claims
1. A method of producing at least one specific pathogen free (SPF)
non-human animal, the method comprising: (a) selecting a surviving
animal in an environment comprising at least one pathogen that is
capable of infecting and/or killing the animal; (b) administration
of a fusion protein to the surviving animal wherein the fusion
protein comprises at least one polypeptide B which is a Type 1
Ribosome Inactivating Protein (RIP) or fragment thereof; and (i) at
least one polypeptide A which is an Antimicrobial peptide; and/or
(ii) at least one polypeptide C which is a Cationic AntiMicrobial
Peptide (CAP) or fragment thereof; and (c) resulting surviving
animal is the SPF non-human animal.
2. The method according to claim 1, further comprising a step of
confirming that the surviving animal from step (a) expresses at
least one marker of a pathogen resistant gene before the
administration of the fusion protein of step (b).
3. The method according to claim 1, wherein the surviving animal is
at least one animal selectively bred for growth.
4. The method according to claim 1, wherein the presence of the SPF
non-human animal in step (c) is confirmed using at least one of the
methods selected from the group consisting of PCR, ELISA, LAMP and
RT-PCR.
5. The method according claim 1, wherein the non-human animal is at
least one aquatic animal.
6. The method according to claim 5, wherein the aquatic animal is
at least one crustacean.
7. (canceled)
8. The method according to claim 2, wherein the marker is selected
from the group consisting of pmAV, c-type lectin, haemocyanin,
beta-integrin, syntenin, alpha-2-macroglobulin, LPS-binding
protein, beta-glucan binding protein, catalase gene, Ras-related
nuclear protein, caspace-3 like gene, calreticulin, Rab GTPase
gene, and Mg-SOD gene.
9. The method according to claim 1, wherein the administration is
by oral delivery.
10. The method according to claim 1, wherein the fusion protein is
administered with food.
11. The method according to claim 1, wherein the SPF non-human
animal is free from at least one pathogen selected from the group
consisting of Avian influenza viruses, Lymphoid Leukosis, Visceral
Leukosis (Marek's Disease), Quail Bronchitis viruses, Newcastle
disease viruses, infectious bronchitis viruses, infectious Bursal
disease viruses, rhinoviruses, echoviruses, equine encephalitis
viruses, coronaviruses, vesicular stomatitis viruses, rabies
viruses, ebola viruses, parainfluenza viruses, Hanta viruses, bunga
viruses, phleboviruses and Nairo viruses, hemorrhagic fever
viruses, reoviruses, orbiviurses and rotaviruses, parvoviruses,
papilloma viruses, polyoma viruses, adenoviruses, Aquabirnaviruses,
Betanoda viruses, Salmonid alphaviruses, Epizotic Hematopoietic
necrosis viruses, Infectious salmon anemia viruses (ISAV), Nervous
necrosis viruses, Abalone Viral ganglioneuritis, Abalone
Herpes-like viruses, variola viruses, vaccinia viruses, pox
viruses, African swine fever virus, Iridovirus, Infectious Salmonid
Anaemia (ISA), White Spot Syndrome Virus (WSSV), Hepatopancreactic
parvo-like virus (HPV), Monodon Baculo virus (MBV), Infectious
Hypodermal and Hematopoietic Necrosis Virus (IHHNV), Yellow Head
Virus (YHV), Taura syndrome virus (TSV), Gill-associated virus
(GAV), Laem-Singh Virus (LSNV), Infectious Myonecrosis Virus
(IMNV), Mourilyan virus (MoV), Koi herpesvirus 1 (KHV 1), KHV2,
KHV3, viral nervous necrosis (VNN), infectious pancreatic necrosis
virus (IPNV), channel catfish virus (CCV), fish lymphocystis
disease virus (FLDV), infectious hematopoietic necrosis virus
(IHNV) and viral hemorrhagic septicemia virus (VHSV), AMAV, swine
hepatitis E virus, Circoviruses, Herpesviruses, Porcine
cytomegalovirus, pseudorabies virus, Feline Panleukopenia virus
(FPV), Feline herpesvirus, Feline calicivirus, Feline Leukemia
Virus (FeLV), Feline Immunodeficiency Virus (FIV), Rabies virus,
canine parvovirus, canine coronavirus, canine distemper virus,
canine influenza, canine hepatitis virus, canine herpesvirus, a
virus that causes pseudorabies, canine minute virus and a
bacteriophage.
12. The method according to claim 11, wherein the bacteriophage is
selected from a group consisting of Myoviridae, Siphoviridae,
Podoviridae, Lipothrixviridae, Rudiviridae, Ampullaviridae,
Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae,
Fuselloviridae, Globuloviridae, Guttavirus, Inoviridae,
Leviviridae, Microviridae, Plasmaviridae, Tectiviridae and the
like. In particular, the phage may be Lambda phage
(.lamda.phage)-lysogen (.lamda.phage), T2 phage, T4 phage, T7
phage, T12 phage, R17 phage, M13 phage, MS2 phage, G4 phage, P1
phage, Enterobacteria phage P2, P4 phage, Phi X 174 phage, N4
phage, Pseudomonas phage .PHI.6, .PHI.29 phage, 186 phage and the
like.
13. The method according to claim 1, wherein the polypeptide A is a
defensin and selected from the group consisting of alpha, beta,
theta, a member of the big defensins protein family, an analogue,
and a fragment thereof.
14. The method according to claim 1, wherein the fusion protein
comprises the structure A-B-C, A-C-B, C-A-B, C-B-A, B-A-C, B-C-A,
A-B-C-C, A-B, B-A, B-C, C-B, C-B-C, or C-C-B-C-C.
15. The method according to claim 1, wherein the fusion protein
comprises polypeptides A, B and C.
16. The method according to claim 1, further comprising at least
one linker peptide between each of the polypeptides A, B and/or
C.
17. The method according to claim 16, wherein the linker peptide
has SEQ ID NO: 3 or 35.
18. The method according to claim 1, wherein polypeptide A is: (a)
a theta defensin selected from the group consisting of Rhesus
minidefensin (RTD-1), RTD-2, RTD-3, Retrocyclin-1, Retrocyclin-2,
Retrocyclin-3, synthetic retrocyclin congener RC100, RC101, RC102,
RC103, RC104, RC105, RC106, RC107, RC108, RC110, RC111, RC112,
RC113 and RC114; or (b) an alpha-defensin selected from the group
consisting of human neutrophil protein 1 (HNP-1), HNP-2, HNP-3,
HNP-4, Human defensin 5 and Human defensin 6, an analogue, or a
fragment thereof; or (c) a beta-defensin selected from the group
consisting of DEFB 1, DEFB 4A, DEFB 4B, DEFB 103A, DEFB 103B, DEFB
104A, DEFB 104B, DEFB 105A, DEFB 105B, DEFB 106A, DEFB 106B, DEFB
107A, DEFB 107B, DEFB 108B, DEFB108 P1-4, DEFB 109 P1, DEFB 109
P1B, DEFB 109 P2-3, DEFB 110, DEFB 112-119 and DEFB 121-136.
19. (canceled)
20. (canceled)
21. The method according to claim 1, wherein the Type 1 RIP
(polypeptide B) is selected from the group consisting of Ebulitins,
Nigritins, Amarandins, Amaranthus antiviral/RIP, Amaranthin,
Atriplex patens RIP, Beta vulgaris RIP, .beta.-vulgin, Celosia
cristata RIP, Chenopodium album RIP, CAP30B, Spinacea oleracea RIP,
Quinqueginsin, Asparins, Agrostin, Dianthins, DAPs, Dianthus
chinensis', Lychnin, Petroglaucin, Petrograndin, Saponaria
ocymoides RIP, Vacuolas saporin, Saporins, Vaccaria hispanica RIP,
Benincasins, Hispin, Byrodins, Colocins, Cucumis figarei RIP,
Melonin, C. moschata RIP, Cucurmo sin, Moschatins, Pepocin,
Gynostemmin, Gynostemma pentaphyllum RIP, Gypsophilin, Lagenin,
Luffaculin, Luffangulin, Luffin, MORs, Momordin II, Momorcharins,
Momorcochin, Momorcochin-S, Sechiumin, Momorgrosvin, Trichoanguin,
Kirilowin, .alpha.-trichosanthin, TAP-29, Trichokirin,
Trichomislin, Trichosanthin, Karasurin, Trichomaglin, Trichobakin,
Crotin, Euserratin Antiviral Protein GAP-31, Gelonin, Hura
crepitans RIP, Curcin, Jathropa curcas RIP, Mapalmin, Manutins,
.alpha.-pisavin, Charibdin, Hyacinthus orientalis RIP, Musarmin,
Iris hollandica RIP, Cleroendrum aculeatum RIP, CIPs),Crip-31,
Bouganin, Bougainvilla spectbilis RIP, Bougainvillea.times.buttiana
Antiviral protein 1 (BBAP1), Malic enzymes, MAP-S, pokeweed
antiviral proteins (PAP), PD-SI, DP-S2, Dodecandrin, PIP, PIP2,
Phytolacca octandra anti-viral proteins, Hordeum vulgare RIP's,
Hordeum vulgare sub sp. Vulgare Translational inhibitor II, Secale
cereale RIP, Tritin, Zea diploperemis RIPs, Malus.times.domestica
RIP, Momordica Anti-HIV Protein, Gelonium multiflorum, Mirabilis
expansa 1, phage MU1, betavulgin (Bvg), curcin 2, saporin 6, Maize
RIP (B-32), Tobacco RIP (TRIP), Beetins, Mirabilis antiviral
protein (MAP), Trichosanthin (TCS), luffins, Momorcharins,
Ocymoidin, Bryodin, Pepopsin, .beta.-trichosanthin, Camphorin, YLP,
Insularin, Barley RIP, Tritins, Lamjarin, and Volvariella volvacea
RIP.
22. The method according to claim 1, wherein the CAP (polypeptide
C) is selected from the group consisting of Cyclotides, Siamycins,
NP-06, Gramicidin A, Circulins, Kalatas, Ginkbilobin,
Alpha-Basrubin, Lunatusin, Sesquin, Tricyclon A, Cycloviolacins,
Polyphemusins, hfl-B5, Protegrins (Pig Cathelicidin), Rat
Defensins, Human .beta.-defensins, Temporins, Caerins, Ranatuerins,
Reptile Defensin, Piscidins, Lactoferricin B, Rabbit Neutrophils,
Rabbit .alpha.-Defensin, Retrocyclins, Human .alpha.-Defensins,
Human .beta.-defensin III (HBD3), Rhesus minidefensin
(RTD-1,.theta.-defensin), rhesus .theta.-defensins, Human
neutrophil peptides, Cecropin As, Melittin, EP5-1, Magainin 2s,
hybrid (CE-MA), hepcidin TH1-5, Epinecidin-1, Indolicidin,
Cathelicidin-4, LL-37 Cathelicidin, Dermaseptins, Maximins,
Brevinins, Ranatuerins, Esculentins, Maculatin 1.3, Maximin H5 and
Piscidins, Mundticin KS Enterocin CRL-35, Lunatusin, FK-13 (GI-20
is a derivative), Tachyplesins, Alpha-MSH, Antiviral protein Y3,
Palustrin-3AR, Ponericin L2, Spinigerin, Melectin, Clavanin B, Cow
cathelicidins, Guinea pig cathelicidin CAP11, Sakacin 5X,
Plectasin, Fungal Defensin, GLK-19, lactoferrin (Lf) peptide 2,
Alloferon 1, Uperin 3.6, Dahlein 5.6, Ascaphin-8, Human Histatin 5,
Guineapig neutrophils, Mytilins, EP5-1, Hexapeptide (synthetic)
Corticostatin IV Rabbit Neutrophil 2, Aureins, Latarcin, Plectasin,
Cycloviolins, Vary Peptide E, Palicourein, VHL-1, and Buforins.
23. The method according to claim 1, wherein: (a) the Type 1 RIP is
MAP30, the CAP is Dermaseptin 1 and the polypeptide A is
Retrocyclin 101; or (b) the Type 1 RIP is MAP30, the CAP is
Alloferonl and the polypeptide A is Tachyplesin; or (c) the Type 1
RIP is MAP30, the CAP is Mytillin C10C and the polypeptide A is
AVBD103; or (d) the Type 1 RIP is GAP31, the CAP Dermaseptin1 and
the polypeptide A Retrocyclin 101.
24. The method according to claim 23, wherein the fusion protein in
(a) comprises the amino acid sequence SEQ ID NO: 1; in (b)
comprises the amino acid sequence SEQ ID NO: 34; in (c) comprises
the amino acid sequence SEQ ID NO: 28; and in (d) comprises the
amino acid sequence SEQ ID NO: 36.
25-30. (canceled)
31. A method of producing at least one specific pathogen resistant
(SPR) non-human animal, the method comprising: (a) producing a
specific pathogen free animal according to any one of claims 1-30,
(b) selective breeding of a male and female SPF non-human animal to
produce a SPR non-human animal offspring.
32. A specific pathogen free non-human animal according to claim
1.
33. A specific pathogen resistant non-human animal according to
claim 31.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods of producing
specific pathogen free and/or specific pathogen resistant
animals.
BACKGROUND TO THE INVENTION
[0002] Specific pathogen free (SPF) non-human animals are essential
for research purposes and to maintain standardised health and
farming. Technological advances in the past decade have led to more
sensitive research and commercialization outcomes that are
recognizably affected by the presence of unwanted microorganisms,
especially viruses. Accordingly, there is a need to produce/farm
animals which are free from these unwanted microorganisms (i.e. SPF
animals).
[0003] Diseases are the bane of farmers everywhere, costing farmers
large amounts of money annually. When one animal is affected by a
disease, most animals in the vicinity or within the same farm will
also be infected causing an epizootic that may be both dangerous to
the health of the human being and/or costing the human being lots
of money. Many problems related to diseases are preventable by
exercising common sense and science-based animal-rearing
strategies. One of these methods is by using SPF non-human animals.
For example, Early. Mortality Syndrome (EMS) also known as Acute
Hepatopancreatic Necrosis Syndrome (AHPNS) in shrimp typically
manifests in the first 10-40 days after stocking in ponds. It began
in China in 2009, spread to Vietnam in 2010, to Malaysia in 2011
and then to Thailand in 2012 with global losses exceeding USD1
billion annually. Before the recent EMS epizootic, the only other
shrimp virus capable of causing losses exceeding USD1 billion
annually was the White Spot Syndrome Virus (WSSV). The pathogen
responsible for EMS has recently been identified as a strain of
Vibrio parahaemolyticus bacteria that has been transferred a toxic
gene via a specific bacteriophage.
[0004] A reason why SPF animals have become extremely important for
example in aquaculture is because it is now commonplace to farm
alien species that are not endemic to a particular nation and
therefore, to prevent introduction of new microorganisms to a
place, use of SPF animals are required. One example is the use of
the fast growing shrimp species Penaeus vannamei which although
originally of Latin American origin, is now farmed in almost every
nation where shrimp farming is a major aquaculture activity,
replacing the slower growing Tiger Shrimp Penaeus monodon. For P.
vannamei, under commercial conditions in Asian earthen ponds,
typical growth rates of 1.0-1.5 g/wk (with 80-90 percent survival)
are common in the high-density pond system (60-150/m.sup.2)
currently in use in Thailand and Indonesia. In contrast, the growth
(and survival) rate of P. monodon has been declining in recent
years from 1.2 to 1 g/wk (and 45 percent to 55 percent survival)
over the last few years in Thailand. In order to increase the
survival rate of at least the P. monodon, use of SPF versions will
help.
[0005] Standard practices for the production of SPF animals for
example in aquaculture use has been described as long back as 1994
and little has changed since in terms of the actual process. The
main objective has been to provide disease-free fry, fingerlings
and post larvae to aquaculture farms to reduce the risk of disease
introduction causing widespread epizootics.
[0006] Genuine SPF shrimp, by present conventions, are those which
are produced from bio secure facilities, have been repeatedly
examined, tested and found free of specified pathogens using
intensive surveillance protocols and molecular methods, and
originate from brood stock developed with strict founder population
development protocols. These founder populations are generated by
extensive quarantine procedures that result in SPF F1 generations
derived from wild parents. The history of domestication programmes
in various countries, in that such stocks may have been
deliberately in-bred and consists entirely of siblings. This means
that future generations of animals based only on such lines will
probably lead to inbreeding within a few generations. Such
inbreeding has been noted in stocks of P. stylirostris bred in
Tahiti for 22 generations. It has also been noted in captive stocks
of P. vannamei, which were characterized by a diminished ability to
tolerate Taura Syndrome virus (TSV) challenges compared to a more
diverse, heterozygous wild control population.
[0007] Accordingly, although SPF animals have their advantages,
producing them is a time-consuming process that may result in other
problems such as inbreeding. Further the potential drawback of SPF
animals is that they are only SPF for the specific diseases for
which they have been checked. However, there is yet to be any SPF
source of EMS-free brood stock shrimp or post larvae available
globally.
[0008] There is thus a need in the art for not only a quicker way
of obtaining a SPF animal but also a simpler method of producing
Specific Pathogen Resistant (SPR) animals.
[0009] SPR describes a genetic trait of a shrimp that confers some
resistance against one specific pathogen. SPR shrimp usually result
from a specific breeding programme designed to increase resistance
to a particular virus. SPF and SPR are independent characteristics.
Not all SPR shrimp are SPF and vice versa. A selective breeding
programme for P. vannamei was initiated in 1995 in the Oceanic
Institute in Hawaii. Original work was based on a selection index
weighted equally for growth and TSV resistance (the major disease
problem in the Americas at that time). Confirmation that growth and
survival (to TSV challenge) responded well to selection was
obtained, but there appeared to be a negative genetic correlation
between these traits. Further investigation revealed that the
shrimp selected only for growth were 21 percent larger than
unselected shrimp (24 vs. 20 g) after one generation, with a
realized heritability (h2) of 1. Females were 12.7 percent larger
than males at about 22 g, but it was not possible to select for a
higher percentage of females. Meanwhile, shrimp selected on an
index weighted 70 percent for TSV resistance and 30 percent for
growth showed an 18 percent increase in survival to a TSV challenge
(46 vs. 39 percent) after one generation, with a realized
heritability (h2) of 0.28. However, selected shrimp were 5 percent
smaller than control shrimp, revealing a negative genetic
correlation between mean family growth and mean family survival to
a TSV challenge. This negative correlation between growth and
disease resistance must therefore be taken into account when
developing breeding plans for these shrimp. Taura Syndrome Virus or
TSV can cause significant losses in farms stocked with P. vannamei
and can be transmitted easily through insect or avian vectors
between ponds. Because of this, the use of TSV-resistant (TSV-SPR)
strains combined with biosecurity measures to reduce infections
with other viruses such as WSSV, IHHNV and YHV could greatly assist
the development of the new culture industry for P. vannamei in
Asia. Such a protocol was adopted by the United States of America
industry that, as a result, has seen a 50 percent growth rate per
year over the last few years.
[0010] In view of the above, SPF and SPR are both essential and
improved methods of producing and breeding them is needed.
SUMMARY OF THE INVENTION
[0011] The present invention is defined in the appended independent
claims. Some optional features of the present invention are defined
in the appended dependent claims.
[0012] According to one aspect of the present invention, there is
provided a method of producing at least one specific pathogen free
(SPF) non-human animal, the method comprising: [0013] (a) selecting
a surviving animal in an environment comprising at least one
pathogen that is capable of infecting and/or killing the animal;
[0014] (b) administration of a fusion protein to the surviving
animal wherein the fusion protein comprises at least one
polypeptide B which is a Type 1 Ribosome Inactivating Protein (RIP)
or fragment thereof; and [0015] (i) at least one polypeptide A
which is an Antimicrobial peptide; and/or [0016] (ii) at least one
polypeptide C which is a Cationic Antimicrobial Peptide (CAP) or
fragment thereof; and [0017] (c) resulting surviving animal is the
SPF non-human animal. Step (c) may be confirmed using
conventionally accepted molecular methods that show the absence of
the pathogen in question.
[0018] In another aspect of the present invention, there is
provided a method of producing at least one specific pathogen
resistant (SPR) non-human animal, the method comprising: [0019] (a)
producing a specific pathogen free animal according to any method
of the present invention; and [0020] (b) selective breeding of a
male and female SPF non-human animal to produce a SPR non-human
animal offspring. Step (b) may occur at the first, second, third,
forth, fifth or tenth generation in the procedure of selective
breeding. In particular, step (b) may eventually occur.
[0021] According to a further aspect of the present invention,
there is provided a specific pathogen free or resistant non-human
animal produced by any method of the present invention.
[0022] As will be apparent from the following description,
preferred embodiments of the present invention allow for a fusion
protein with an optimal effectiveness with a broad spectrum therapy
and/or allowing oral delivery of the protein as some of the several
applications.
BRIEF DESCRIPTION OF THE FIGURES
[0023] Preferred embodiments of the fusion protein will now be
described by way of example with reference to the accompanying
figures in which:
[0024] FIG. 1 is a translation map of RetroMAD1 (SEQ ID NO:1 and
SEQ ID NO:2).
[0025] FIG. 2 has two photos of gels showing A) Time course
expression and B) Solubility of RetroMAD1 expression in E. Coli
BL21(DE3) cells. Cells harbouring pRMD were harvested before
induction (Oh), and after induction for 1 h, 2 h and 3 h represents
the pellet phase, the hours with asterisk (*) represents the
supernatant phase. Proteins were analysed on a 15% SDS-PAGE. M:
PageRuler.TM. Protein Ladder Fermentas, U: uninduced, IND: induced
and IB: purified inclusion bodies. Arrow indicates E. coli produced
RetroMAD1 (41.2 kDa).
[0026] FIG. 3 is a photo of an agarose gel showing the PCR products
in particular, the expected band of 441 by confirming the absence
of the virus in the RetroMAD1 treated prawns.
[0027] FIG. 4 shows the experimental set-up of Example 3 to test
the effects of RetroMAD1 on WSSV.
[0028] FIG. 5 are graphs showing the results that RetroMAD1 treated
prawns survived for a longer period of time compared to the control
(i.e. WSSV infected prawns).
[0029] FIG. 6 are gel images of showing the stability of fusion
proteins, RetroMAD1, RetroGAD1, Amatilin and Tamapal1: A1 and A2
are RetroMAD1 subjected to temperature fluctuations; B1 and B2 are
RetroGAD1 subjected to temperatures; C1 and C2 are Amatilin
subjected to temperature fluctuations; D1 and D2 are Tamapal1
subjected to temperature fluctuations. Protein Ladder is the marker
for protein size; Control is untreated drug; T1-4 are the different
temperature fluctuations (as shown in Table 6) BME is
2.times..beta.-mercaptoethanol, the samples are loaded with (+) or
without (-) BME.
[0030] FIG. 7 is a graph showing the percentage of viral reduction
caused by Amatilin, RetroGAD1 and Tamapal1 exposed to various
temperature fluctuations in simultaneous treatment determined by
PCR.
[0031] FIG. 8A-D are graphs showing concentration of RetroMAD1
(.mu.g/ml) (A), RetroGAD1 (.mu.g/ml) (B), Amatilin (.mu.g/ml) (C),
Tamapal1 (.mu.g/ml) (D) leached out against Time (minutes)
[0032] FIG. 9 is a graph showing concentration of RetroMAD1 in
hepatopancreas, tail muscle, faeces and control against time in a
short-term pharmacokinetics study
[0033] FIG. 10 is a graph showing concentration of RetroMAD1 in
hepatopancreas, tail muscle, faeces and control against time in a
long-term pharmacokinetics study
[0034] FIGS. 11A and B is an image of plates showing the
anti-bacterial activity of amatilin against V. cholera (A) and V.
parahemolyticus (B).(Plate: 1. 322.5 .mu.g/ml, 2. 161.25 .mu.g/ml,
3. 80.63 .mu.g/ml, 4. 40.31 .mu.g/ml, 5. 20.16 .mu.pg/ml, 6. 10.08
.mu.g/ml, 7. 5.04 .mu.g/ml, 8. 2.52 .mu.g/ml, 9. Untreated)
[0035] FIG. 12 is a graph showing the percentage of viral reduction
caused by Amatilin, RetroGAD1, RetroMAD1 and Tamapal1 in
simultaneous treatment at 72 h determined by PCR.
[0036] FIG. 13 A-C is a graph showing the percentage of viral
reduction caused by drugs (A: Amatilin; B: RetroGAD1, C: Tamapal1)
incubated at different temperatures for 1, 7 and 30 days in
simultaneous treatment determined by PCR. (* Thermostability was
not tested for 50.degree. C. for 30 days incubation)
[0037] FIG. 14 is a schematic diagram showing the Supercritical
Fluid Drying (SCFD) Process
[0038] FIG. 15 is a Scanning Electron Microscope (SEM) image of
RetroMAD1 crystals
[0039] FIG. 16 is a graph showing the percentage of viral reduction
caused by RetroMAD1 micronized powder in simultaneous treatment
determined by PCR.
DETAILED DESCRIPTION OF THE INVENTION
[0040] For convenience, certain terms employed in the
specification, examples and appended claims are collected here.
[0041] The term "adjuvant", as used in the context of the invention
refers to an immunological adjuvant. By this, an adjuvant is meant
to be a compound that is able to enhance or facilitate the immune
system's response to the ingredient in question, thereby inducing
an immune response or series of immune responses in the subject.
The adjuvant can facilitate the effect of the therapeutic
composition by forming depots (prolonging the half-life of the
ingredient), provide additional T-cell help and stimulate cytokine
production. Facilitation of antigen survival and unspecific
stimulation by adjuvants may, in some cases, be required if the
antigenic molecule are only weakly antigenic or only exerts weak to
moderate interactions with compounds, molecules, or cells of the
immune system.
[0042] The term "analogue" as used in the context of the invention
refers to a peptide that may be modified by varying the amino acid
sequence to comprise one or more naturally-occurring and/or
non-naturally-occurring amino acids, provided that the peptide
analogue is capable of reducing or preventing growth of a tumour or
cancer. For example, the term "analogue" encompasses an inhibitory
peptide comprising one or more conservative amino acid changes. The
term "analogue" also encompasses a peptide comprising, for example,
one or more D-amino acids. Such an analogue has the characteristic
of, for example, protease resistance. Analogues also include
peptidomimetics, e.g., in which one or more peptide bonds have been
modified. Preferred analogues include an analogues of a peptide as
described according to any embodiment here comprising one or more
non-naturally-occurring amino acid analogues.
[0043] The term "comprising" as used in the context of the
invention refers to where the various components, ingredients, or
steps, can be conjointly employed in practicing the present
invention. Accordingly, the term "comprising" encompasses the more
restrictive terms "consisting essentially of" and "consisting of."
With the term "consisting essentially of" it is understood that the
epitope/antigen of the present invention "substantially" comprises
the indicated sequence as "essential" element. Additional sequences
may be included at the 5' end and/or at the 3' end. Accordingly, a
polypeptide "consisting essentially of" sequence X will be novel in
view of a known polypeptide accidentally comprising the sequence X.
With the term "consisting of" it is understood that the
polypeptide, polynucleotide and/or antigen according to the
invention corresponds to at least one of the indicated sequence
(for example a specific sequence indicated with a SEQ ID Number or
a homologous sequence or fragment thereof).
[0044] The term "derivative" as used in the context of the
invention includes e.g., a fragment or processed form of the stated
peptide, a variant or mutant comprising one or more amino acid
substitutions, deletions of additions relative to the stated
peptide, a fusion protein comprising the stated peptide or a
peptide comprising one or more additional non-peptide components
relative to the stated peptide e.g., a chemical component, e.g.,
polyethylene glycol (PEG). The term "derivative" also encompasses
polypeptides comprising the fusion protein according to the
invention. For example, the polypeptide comprises a label, such as,
for example, an epitope, e.g., a FLAG epitope or a V5 epitope or an
HA epitope. For example, the epitope is a FLAG epitope. Such a tag
is useful for, for example, purifying the polypeptide. A preferred
derivative of an antitumour or anticancer fusion protein of the
invention has enhanced stability. For example, a cleavage site of a
protease active in a subject to which a fusion protein is to be
administered is mutated and/or deleted to produce a stable
derivative of an antitumour or anticancer fusion protein of the
invention. The term "derivative" also encompasses a derivatized
peptide, such as, for example, a peptide modified to contain one or
more-chemical moieties other than an amino acid. The chemical
moiety may be linked covalently to the peptide e.g., via an amino
terminal amino acid residue, a carboxy terminal amino acid residue,
or at an internal amino acid residue. Such modifications include
the addition of a protective or capping group on a reactive moiety
in the peptide, addition of a detectable label, and other changes
that do not adversely destroy the activity of the peptide
compound.
[0045] Accordingly, acceptable amino acid substitutions are
generally therefore based on the relative similarity of the amino
acid side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. Exemplary substitutions
which take several of the foregoing characteristics into
consideration are well known to those of skill in the art and
include: arginine and lysine; glutamate and aspartate; serine and
threonine; glutamine and asparagine; and valine, leucine and
isoleucine. The isolated peptides of the present invention can be
prepared in a number of suitable ways known in the art including
typical chemical synthesis processes to prepare a sequence of
polypeptides.
[0046] The term "fragment" as used in the context of the invention
refers to an incomplete or isolated portion of the full sequence of
the fusion protein according to any aspect of the present invention
which comprises the active site(s) that confers the sequence with
the characteristics and function of the protein. In particular, it
may be shorter by at least one amino acid. For example a fragment
of the fusion protein according to the present invention comprises
the active site(s) that enable the protein to recognise an aberrant
cell such as a tumour cell or cancer cell. The fragment may at
least be 10 amino acids in length. For example, a non-limiting
fragment of RIP may at least comprise the core or the bioactive
site of the RIP which may be approximately 5 kDa in size.
[0047] The term "fusion protein(s)" as used in the context of the
invention refers to proteins created through the joining of two or
more genes, which originally coded for separate proteins.
Translation of this fusion gene results in a single polypeptide
with functional properties derived from each of the original
proteins. Recombinant fusion proteins are created artificially by
recombinant DNA technology for use in biological research or
therapeutics. For example, the fusion protein according to any
aspect of the present invention may comprise a polypeptide B; and a
polypeptide C which is a CAP. The fusion protein may have antiviral
properties. The fusion protein according to any aspect of the
present invention may further comprise a polypeptide A. Each
individual part and/or the whole the fusion protein may have
antiviral properties. For example, polypeptide A, B, and/or C may
have anticancer properties. As a whole A-B-C may have antiviral
properties. The structure of the fusion protein may be A-B-C,
A-C-B, C-A-B, C-B-A, B-A-C, B-C-A, A-B-C-C, A-B, B-C, B-C-C,
C-C-B-C-C, or C-B-C. In particular, the fusion protein may comprise
dimers and/or tandem repeats. More in particular, the structure of
the fusion protein according to any aspect of the present invention
may be repeats of the structure mentioned above. For example, the
structure may be A-A-B-C-C, C-C-B-C-C, A-A-B-A-A and the like. The
polypeptide A, B or C in each fusion protein may be the same
protein or may be a different protein when repeated. Polypeptide A
may be theta defensin, an analogue, or a fragment thereof. A fusion
protein according to the present invention may comprise the
sequence of SEQ ID NO:1, a variant, derivative or fragment thereof.
The term "RetroMAD1" is used in the present invention to refer to a
fusion protein with the structure A-B-C and with amino acid
sequence SEQ ID NO:1. In particular, in RetroMAD1 polypeptide A may
be Retrocyclin 101, polypeptide B may be MAP30 and polypeptide C
may be Dermaseptin 1. These peptides may be directly fused to one
another or connected to one another by a linker peptide.
[0048] The term "linker peptide", as used in the context of the
invention is used interchangeably with the term "linker" herein. A
linker peptide is a peptide that covalently or non-covalently
connects two or more molecules or peptides, thereby creating a
larger complex consisting of all molecules or peptides including
the linker peptide. A non-limiting example of a linker peptide may
be SEQ ID NO:3 and/or SEQ ID NO:27.
[0049] The term "pathogen" as used in the context of the invention
may refer to any disease-producing agent, especially a virus,
bacterium, or other microorganism. A virus may be selected from the
group consisting of cytomegalovirus (CMV), Epstein-Barr virus
(EBV), varicella zoster virus (VZV), HSV-1, HSV-2, HSV-6, BK-virus,
influenza viruses, respiratory syncytial virus (RSV); human
immunodeficiency virus (HIV), hepatitis A, B or C (HBV), polio
viruses, enteroviruses, human coxsackie viruses, rhinoviruses,
echoviruses, equine encephalitis viruses, rubella viruses, dengue
viruses, encephalitis viruses, yellow fever, coronaviruses,
vesicular stomatitis viruses, rabies viruses, ebola viruses,
parainfluenza viruses, mumps virus, measles virus, Hanta viruses,
bunga viruses, phleboviruses and Nairo viruses, hemorrhagic fever
viruses, reoviruses, orbiviurses and rotaviruses, parvoviruses,
papilloma viruses, polyoma viruses, adenoviruses, herpes simplex
virus (HSV) 1 and HSV-2, varicella zoster virus, variola viruses,
vaccinia viruses, pox viruses, African swine fever virus,
Iridovirus, Infectious Salmonid Anaemia (ISA), White Spot Syndrome
Virus (WSSV), Hepatopancreactic parvo-like virus (HPV), Monodon
Baculo virus (MBV), Infectious Hypodermal and Hematopoietic
Necrosis Virus (IHHNV), Yellow Head Virus (YHV), Taura syndrome
virus (TSV), Gill-associated virus (GAV), Laem-Singh Virus (LSNV),
Infectious Myonecrosis Virus (IMNV), Mourilyan virus (MoV), Koi
herpesvirus 1 (KHV 1), KHV2, KHV3, viral nervous necrosis (VNN),
infectious pancreatic necrosis virus (IPNV), channel catfish virus
(CCV), fish lymphocystis disease virus (FLDV), infectious
hematopoietic necrosis virus (IHNV) and viral hemorrhagic
septicemia virus (VHSV), AVG, AMAV, swine hepatitis E virus,
Circoviruses, Herpesviruses, Porcine cytomegalovirus, pseudorabies
virus, Feline Panleukopenia virus (FPV), Feline herpesvirus, Feline
calicivirus, Feline Leukemia Virus (FeLV), Feline Immunodeficiency
Virus (FIV), Rabies virus, canine parvovirus, canine coronavirus,
canine distemper virus, canine influenza, canine hepatitis virus,
canine herpesvirus, a virus that causes pseudorabies, canine minute
virus and the like.
[0050] In particular, the viruses may only be viruses that are
capable of infecting a non-human animal. The virus may be selected
from the group consisting of Avian influenza viruses, Lymphoid
Leukosis, Visceral Leukosis (Marek's Disease), Quail Bronchitis
viruses, Newcastle disease viruses, infectious bronchitis viruses,
infectious Bursal disease viruses, rhinoviruses, echoviruses,
equine encephalitis viruses, coronaviruses, vesicular stomatitis
viruses, rabies viruses, ebola viruses, parainfluenza viruses,
Hanta viruses, bunga viruses, phleboviruses and Nairo viruses,
hemorrhagic fever viruses, reoviruses, orbiviurses and rotaviruses,
parvoviruses, papilloma viruses, polyoma viruses, adenoviruses,
Aquabirnaviruses, Betanoda viruses, Salmonid alphaviruses, Epizotic
Hematopoietic necrosis viruses, Infectious salmon anemia viruses
(ISAV), Nervous necrosis viruses, Abalone Viral ganglioneuritis,
Abalone Herpes-like viruses, variola viruses, vaccinia viruses, pox
viruses, African swine fever virus, Iridovirus, Infectious Salmonid
Anaemia (ISA), White Spot Syndrome Virus (WSSV), Hepatopancreactic
parvo-like virus (HPV), Monodon Baculo virus (MBV), Infectious
Hypodermal and Hematopoietic Necrosis Virus (IHHNV), Yellow Head
Virus (YHV), Taura syndrome virus (TSV), Gill-associated virus
(GAV), Laem-Singh Virus (LSNV), Infectious Myonecrosis Virus
(IMNV), Mourilyan virus (MoV), Koi herpesvirus 1 (KHV 1), KHV2,
KHV3, viral nervous necrosis (VNN), infectious pancreatic necrosis
virus (IPNV), channel catfish virus (CCV), fish lymphocystis
disease virus (FLDV), infectious hematopoietic necrosis virus
(IHNV) and viral hemorrhagic septicemia virus (VHSV), AMAV, swine
hepatitis E virus, Circoviruses, Herpesviruses, Porcine
cytomegalovirus, pseudorabies virus, Feline Panleukopenia virus
(FPV), Feline herpesvirus, Feline calicivirus, Feline Leukemia
Virus (FeLV), Feline Immunodeficiency Virus (FIV), Rabies virus,
canine parvovirus, canine coronavirus, canine distemper virus,
canine influenza, canine hepatitis virus, canine herpesvirus, a
virus that causes pseudorabies, canine minute virus and the
like.
[0051] A virus may include a bacteriophage, also known as a phage
that includes a group of viruses that infect specific bacteria,
usually causing their disintegration or dissolution. A
bacteriophage may be selected from a group consisting of
Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae,
Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae,
Corticoviridae, Cystoviridae, Fuselloviridae, Globuloviridae,
Guttavirus, Inoviridae, Leviviridae, Microviridae, Plasmaviridae,
Tectiviridae and the like. In particular, the phage may be Lambda
phage (.gamma. phage)-lysogen (.lamda. phage), T2 phage, T4 phage,
T7 phage, T12 phage, R17 phage, M13 phage, MS2 phage, G4 phage, P1
phage, Enterobacteria phage P2, P4 phage, Phi X 174 phage, N4
phage, Pseudomonas phage .phi.6, .phi.29 phage, 186 phage and the
like.
[0052] A bacteria may include Aeromonas hydrophila, Aeromonas
salmonicida, Aeromonas sobrio, Enterobacter aerogenes, Enterococcus
faecalis, Escherichia coli, Flavobacterium meningosepticum,
Helicobacter pylori, Klebsiella pneumonia, Listeria monocytogenes,
Listonella anguillarum, Methicillin-resistant Staphylococcus
aureus, Micrococcus luteus, Morganella morganii, Pasturella
multocida, Pseudomonas aeruginosa, Salmonella typhimurium,
Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus
haemolyticus, Streptococcus agalactiae, Streptococcus equi,
Streptococcus iniae, Streptococcus uberis, Vibrio alginolyticus,
Vibrio anguillarum, Vibrio cholera, Vibrio damsel, Vibrio
fluvialis, Vibrio furnissi, Vibrio harveyi, Vibrio hollisae, Vibrio
metschnikovii, Vibrio mimicus, Vibrio parahaemolyticus, Vibrio
proteolyticis, Vibrio vulnificus, Vibrio splendidus, Yersinia
ruckeri and the like.
[0053] The term "polypeptide" as used in the context of the
invention may refer to a long, continuous, and unbranched peptide
and may include cyclic polypeptides. Proteins consist of one or
more polypeptides arranged in a biologically functional way and may
often be bound to cofactors, or other proteins. In particular, the
protein according to any aspect of the present invention may be
naturally occurring, de novo and/or synthetic.
[0054] The terms "subject", "patient" and "individual" are used
interchangeably and are used in the context of the invention refers
to either a human or a non-human animal. These terms include
mammals, such as humans, primates, livestock animals (including
bovines, porcines, etc.), companion animals (e.g. canines, felines,
etc) and rodents (e.g. mice and rats). In particular, the subject
is an aquatic animal. The aquatic animal can be any animal, either
vertebrate or invertebrate, which lives in the water for most or
all of its life. The aquatic animal may be an arthropod for example
a Horseshoe crab. In particular, the aquatic animal can be any
crustacean which includes but is not limited to crabs, lobsters,
crayfish, langoustine, shrimp, and prawn. For example, a prawn can
be decapod crustaceans. The term "prawn" can include cold water
prawn, warm water prawn, caridean shrimp, whiteleg shrimp, Atlantic
white shrimp, Indian prawn, banana prawn, tiger prawn and the like.
In another example, the aquatic animal can be any fish, such as,
for example, the Toad fish, zebra fish, Grouper or salmon; any
crustacean such as, for example fiddler crab, or crayfish; or any
cephalopod such as, for example, a squid. The aquatic animal can
also be an amphibian such as, for example, a frog or salamander.
The aquatic animal can be an animal adapted to fresh water,
seawater, or brackish water. Both brackish water and seawater are
saltwater. Brackish water has more salinity than fresh water, but
less than seawater, such as the water in estuaries.
[0055] The term "variant", as used in the context of the invention
can alternatively or additionally be characterised by a certain
degree of sequence identity to the parent polypeptide from which it
is derived. More precisely, a variant in the context of the present
invention exhibits at least 30% sequence identity, in particular at
least 40%, 50%, 60%, 70%, 80% or 90% sequence identity. More in
particular, a variant in the context of the present invention
exhibits at least 95% sequence identity to its parent polypeptide.
The variants of the present invention exhibit the indicated
sequence identity, and preferably the sequence identity is over a
continuous stretch of 100, 150, 200, 300, 315, 320, 330, 340, 344
or more amino acids. The similarity of nucleotide and amino acid
sequences, i.e. the percentage of sequence identity, can be
determined via sequence alignments. Such alignments can be carried
out with several art-known algorithms, preferably with the
mathematical algorithm of Karlin and Altschul (Karlin &
Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with
hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the
CLUSTAL available e.g. on http://www.ebi.ac.uk/Tools/clustalw/.
Preferred parameters used are the default parameters as they are
set on http://www.ebi.ac.uk/Tools/clustalw/ or
http://www.ebi.ac.uk/Tools/clustalw2/index.html. The grade of
sequence identity (sequence matching) may be calculated using e.g.
BLAST, BLAT or BlastZ (or BlastX). Preferably, sequence matching
analysis may be supplemented by established homology mapping
techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19
Suppl 1 :154-162) or Markov random fields. When percentages of
sequence identity are referred to in the present application, these
percentages are calculated in relation to the full length of the
longer sequence, if not specifically indicated otherwise.
[0056] The phrase "Specific pathogen free (SPF) animal" is a
special stock of animals that are kept in specific pathogen free
facilities under rigorous monitoring system, which are subjected to
sensitive and accurate diagnostic methods. The traditional methods
of producing SPF includes the animals being repeatedly bred under
controlled conditions to maintain their freedom from specific
pathogens and the SPF designation itself is tested on a regular
basis over an extended period of time. The SPF animals may not
innately be resistant to the specified pathogens or infections,
although they can possibly be developed as specific pathogen
resistant (SPR) species. They are not produced to provide either
superior genetic stock or improved culturing attributes such as
faster growth. However, these characteristics can be incorporated
into SPF stock to increase their commercial value. The SPF status
of stock animals may be lost once the animals are removed from the
designated facility even if the animals are not infected or develop
any other disease symptoms. The SPF animals may be referred to as
"high health" stock once they are transferred to other
well-established unit with history of disease surveillance.
[0057] A person skilled in the art will appreciate that the present
invention may be practiced without undue experimentation according
to the method given herein. The methods, techniques and chemicals
are as described in the references given or from protocols in
standard biotechnology and molecular biology textbooks.
[0058] In one aspect of the present invention, there is provided a
method of producing at least one specific pathogen free (SPF)
non-human animal, the method comprising: [0059] (a) selecting a
surviving animal in an environment comprising at least one pathogen
that is capable of infecting and/or killing the animal; [0060] (b)
administration of a fusion protein to the surviving animal wherein
the fusion protein comprises at least one polypeptide B which is a
Type 1 Ribosome Inactivating Protein (RIP) or fragment thereof; and
[0061] (i) at least one polypeptide A which is an Antimicrobial
peptide; and/or [0062] (ii) at least one polypeptide C which is a
Cationic Antimicrobial Peptide (CAP) or fragment thereof; and
[0063] (c) resulting surviving animal is the SPF non-human
animal.
[0064] In particular, the specific pathogen free non-human animal
may be considered an "instant specific pathogen free" or ISPF
non-human animal that may be breeding stock indicating that a
"viral clean-up" is possible.
[0065] The "surviving animal" in step (a) may be any animal that
may be capable of enduring the environment with at least one
pathogen thus staying alive in the presence of the pathogen. The
environment may be considered "challenging" allowing selective
breeding to take place thus the surviving animal may be considered
a suitable candidate for SPF and/or SPR.
[0066] The method may further comprise a step of confirming that
the surviving animal from step (a) expresses at least one marker of
a pathogen resistant gene before the administration of the fusion
protein of step (b). These markers may be well known in the art to
be specific the particular animal. In particular, these markers may
be known in the art to be expressed in a particular animal that is
resistant to at least one pathogen. For example, if the animal is a
prawn, the marker may be selected from the group consisting of
pmAV, c-type lectin, haemocyanin, beta-integrin, syntenin,
alpha-2-macroglobulin, LPS-binding protein, beta-glucan binding
protein, catalase gene, Ras-related nuclear protein, caspace-3 like
gene, calreticulin, Rab GTPase gene, Mg-SOD gene and the like.
Similarly, each species of animal may have markers that are
specific to that animal.
[0067] For example, the development of WSSV-resistant (WSSV-SPR)
lines of P. vannamei is recognized a possibility and because WSSV
remains the biggest disease problem in Asian shrimp culture, this
would provide a much-needed impetus for the Asian shrimp culture
industry as a whole. The recent applications of quantitative
genetics to shrimp breeding, including the identification of
various molecular markers (particularly microsatellites) associated
with disease resistance and growth, offer a method through which
the selection of fast-growing, disease resistant strains might soon
become much more efficient. It may also shed some light on
invertebrate antiviral immunity, about which currently nothing is
known. Such disease related markers have already been identified
for IHHNV in P. stylirostris (Hizer S. E. et. al., 2002). The genes
that are up-regulated in shrimp during a WSSV infection has been
reviewed (Liu H. et. al., 2009) and these may now be used as
disease resistance markers for selective breeding.
[0068] The surviving animal in step (a) may be at least one animal
that has been selectively bred for growth prior to carrying out the
method according to any aspect of the present invention. In one
example, the animals bred in the environment comprising at least
one pathogen that is capable of infecting and/or killing the animal
have been pre-selected for growth and/or any other advantageous
trait and may grow at a faster rate than the wild type of the
animal. This may also the SPF animal using any method of the
present invention may be achieved earlier than the methods known in
the art.
[0069] There may be a negative correlation between growth and
disease resistance (Argue B. et. al., 2002) and hence, it may be
best to begin from a population that had been pre-selected for
growth.
[0070] The presence of the SPF non-human animal in step (c) may be
confirmed using any method known in the art. In particular, the
presence of the SPF non-human animal may be confirmed by
determining the presence or absence of the virus in the animal. The
method of determining may be any method known in the art that is
capable of identifying the presence of any genetic material of the
virus in the animal. For example, the method of determining the
presence of at least one SPF non-human animal may be selected from
the group consisting of PCR, ELISA, RT-PCR, LAMP and the like.
[0071] Thus, for example, any shrimp candidate or any aquatic
animal, may be fed any fusion protein according to any aspect of
the present invention along with its feed until it may became PCR
negative for the specific viruses to be checked. If this animal
were from a known SPF-line that had been selected for growth,
preferably over 3 or more generations, and if this animal were to
be grown in pond conditions where viruses were common, the
`survivors` of any resulting epizootic if any, would have a high
probability of carrying resistance genes that may be screened using
PCR, RT-PCR or microarrays and these could be used to selectively
breed a SPR line over time. Microarrays may be used to study shrimp
immune responses under various conditions which is a convenient and
rapid method to screen survivor populations for resistance
genes.
[0072] The SPF animal may free from at least one pathogen selected
from the group consisting of cytomegalovirus (CMV), Epstein-Barr
virus (EBV), varicella zoster virus (VZV), HSV-1, HSV-2, HSV-6,
BK-virus, influenza viruses, respiratory syncytial virus (RSV);
human immunodeficiency virus (HIV), hepatitis A, B or C (HBV),
polio viruses, enteroviruses, human coxsackie viruses,
rhinoviruses, echoviruses, equine encephalitis viruses, rubella
viruses, dengue viruses, encephalitis viruses, yellow fever,
coronaviruses, vesicular stomatitis viruses, rabies viruses, ebola
viruses, parainfluenza viruses, mumps virus, measles virus,
respiratory syncytial virus, Hantaan viruses, bunga viruses,
phleboviruses and Nairo viruses, hemorrhagic fever viruses,
reoviruses, orbiviurses and rotaviruses, parvoviruses, papilloma
viruses, polyoma viruses, adenoviruses, herpes simplex virus (HSV)
1 and HSV-2, varicella zoster virus, variola viruses, vaccinia
viruses, pox viruses, African swine fever virus, WSSV, HPV, MBV,
IHHNV, YHV, TSV, GAV, LSNV, IMNV, MoV, KHV1, KHV2, KHV3, VNN,
pancreatic necrosis virus (IPNV), channel catfish virus (CCV), fish
lymphocystis disease virus (FLDV), hematopoietic necrosis virus
(IHNV) and viral hemorrhagic septicemia virus (VHSV), AVG, AMAV,
swine hepatitis E virus, Circoviruses, Herpesviruses, Porcine
cytomegalovirus, pseudorabies virus, Feline Panleukopenia virus
(FPV), Feline herpesvirus, Feline calicivirus, Feline Leukemia
Virus (FeLV), Feline Immunodeficiency Virus (FIV), Rabies virus,
canine parvovirus, canine coronavirus, canine distemper virus,
canine influenza, canine hepatitis virus, canine herpesvirus, a
virus that causes pseudorabies, and canine minute virus.
[0073] The fusion protein according to any aspect of the present
invention may be an antiviral compound capable of a broad spectrum
of applications and that may be economically produced without any
limitation of raw material supply unlike certain antiviral
compounds known in the art.
[0074] In order to achieve broad-spectrum activity, the fusion
peptide according to any aspect of the present invention may be
able to interfere with viral growth or proliferation in a number of
different pathways. The fusion protein may thus have a
multifunctional ability. An entire new class of antiviral drugs may
thus be produced from the fusion protein according to any aspect of
the present invention. The number of combinations and permutations
that may be obtained from expressed polypeptides A, B, and C as
fusion antiviral proteins potentially numbers in the tens of
thousands.
[0075] In particular, the fusion protein may comprise at least one
formula selected from the group consisting of formulas I-XIX:
A-B-C, Formula I
A-B-C-C, Formula II
A-B, Formula III
A-C-B, Formula IV
C-A-B, Formula V
C-B-A, Formula VI
C-B, Formula VII
B-A-C, Formula VIII
B-A-C-C, Formula IX
B-C-A, Formula X
B-C, Formula XI
B-A, Formula XII
C-C-B-C-C, Formula XIII
C-B-C, Formula XIV
[0076] Polypeptide A may be an antimicrobial peptide. In
particular, polypeptide A may be a viral entry inhibitory protein.
More in particular, polypeptide A may be a defensin, an analogue,
or a fragment thereof. Even more in particular, the defensin may be
an alpha, a beta, theta defensin, and a member of the Big defensins
protein family, an analogue, or a fragment thereof. Polypeptide B
may be Type 1 RIP, or a fragment thereof, polypeptide C may be
Cationic Antimicrobial Peptide (CAP) or a fragment thereof;
and--may be a direct linkage or a linker peptide.
[0077] In particular, the linker peptide may comprise a polypeptide
sequence: [VPXVG].sub.n,(SEQ ID NO:3) wherein X is an unknown or
other amino acid and n is the number of repeats of SEQ ID NO:3 in
each linker peptide. For example, n may be 1, 2, 3, 4 or 5. More in
particular, X in SEQ ID NO:3 is G and n is 2.
[0078] In another example, the linker peptide may be a
glycine-serine linker. In particular, the glycine-serine linker may
have a sequence of [G-G-G-S].sub.n (SEQ ID NO:27).
[0079] In particular, the fusion protein may comprise the formula
I:
A-B-C-
wherein, polypeptide A is a defensin (.alpha., .beta., .theta.) an
analogue, or a fragment thereof. In particular, polypeptide A may
be a theta defensin, an analogue, or a fragment thereof,
polypeptide B may be Type 1 RIP, or a fragment thereof, and
polypeptide C may be CAP, or a fragment thereof and "-" may be a
direct linkage or a linker peptide.
[0080] More in particular, polypeptide A may be fused to
polypeptide B via at least one first linker peptide of SEQ ID NO:
3. Even more in particular, polypeptide A may be fused to
polypeptide B via a peptide of SEQ ID NO: 3, wherein X is G and n
is 2. Polypeptide B may be directly linked to polypeptide C with no
linker peptide in-between. Polypeptide C in formula I may comprise
a second linker peptide on the free end not linked to B. The second
linker peptide may comprise the formula SEQ ID NO: 3. Even more in
particular, in the second linker peptide X is G and n is 2.
[0081] Polypeptide A may be a viral entry inhibitor protein. In
particular, polypeptide A may be a defensin (.alpha., .beta.,
.theta.) an analogue, or a fragment thereof. In particular,
polypeptide A may be a theta defensin of a vertebrate or
invertebrate origin. In particular, theta Defensin may be from a
bacterium, fungus, mammal, amphibian or reptile. The mammal may be
a non-human primate and/or the invertebrate may be a horseshoe crab
and/or an insect. The theta Defensin may be selected from the group
consisting of Rhesus minidefensin (RTD-1), RTD-2, RTD-3,
Retrocyclin-1, Retrocyclin-2, Retrocyclin-3 from Macaca mulatta of
SEQ ID Nos: 7-12 respectively and the like (Tang Y Q, 1999; Leonava
L, 2001; Wang W, 2004).
[0082] The theta Defensin may be synthetic and may be selected from
a group of retrocyclin congeners RC100-RC108 and RC110-RC114 of SEQ
ID NO:13-25 respectively (Cole et. al. 2002: PNAS, V99(4):1813-1818
; Wang et. al. 2003: J. Immunol. 170:4708-4716). The sequences of
Retrocyclin (RC) 100-108 and RC110-RC114 are shown in Table 1a
below.
TABLE-US-00001 TABLE 1A Polypeptide sequences of naturally
occurring and synthetic theta Defensin proteins. SEQ ID NO:
Sequences 7 GFCRCLCRRGVCRCICTR 8 RCLCRRGVCRCLCRRGVC 9
RCICTRGFCRCICTRGFC 10 GICRCICGRGICRCICGR 11 GICRCICGRGICRCICGR 12
RICRCICGRRICRCICGR 13 GICRCICGRGICRCICGR 14 GICRCICGKGICRCICGR 15
GICRCYCGRGICRCICGR 16 GICRCICGRGICRCYCGR 17 GYCRCICGRGICRCICGR 18
GICRCICGRGYCRCICGR 19 GICYCICGRGICRCICGR 20 GICICICGYGICRCICGR 21
GICICICGRGICYCICGR 22 GICICICGRGICYCICGR 23 RGCICRCIGRGCICRCIG 24
RGCICRCIGRGCICRCIG 25 GICRCICGRGICRCICGR 26 GICRCICGKGICRCYCGR
[0083] Polypeptide A may be a beta defensin. In particular,
polypeptide A may be avian beta defensin (AVBD103).
[0084] Alpha defensins for human are HNP 1-4 and Human Defensin
5-6, and alpha defensins of mice, monkeys, rats, rabbits, guinea
pigs, hamster, horse, elephant, baboon, hedgehog, horse,
chimpanzee, orangutan, macaque and marmoset.
[0085] Beta defensins are DEFB 1, DEFB 4A, DEFB 4B, DEFB 103A, DEFB
103B, DEFB 104A, DEFB 1046, DEFB 105A, DEFB 1056, DEFB 106A, DEFB
106B, DEFB 107A, DEFB 107B, DEFB 108B, DEFB108 P1-4, DEFB 109 P1,
DEFB 109 P1B, DEFB 109 P2-3, DEFB 110, DEFB 112-119, DEFB
121-136.
[0086] Big defensins is a diverse family of antimicrobial peptides.
Members of the Big defensins protein family originate from (i)
Amphioxus--Branchiostoma florida and Branchiostoma belcheri; (ii)
Horseshoecrab--Tachypleus tridentatus; (iii) Mussel--Mytilus
galloprovincialis; (iv) Clam--Ruditapes philippinam; and (v)
Oyster--Crassostrea gigas.
[0087] Polypeptide B may be a Type 1 Ribosome Inactivating Protein
selected from the group consisting of Ebulitins, Nigritins,
Amarandins, Amaranthus antiviral/RIP, Amaranthin, Atriplex patens
RIP, Beta vulgaris RIP, .beta.-vulgin, Celosia cristata RIP,
Chenopodium album RIP, CAP30B, Spinacea oleracea RIP,
Quinqueginsin, Asparins, Agrostin, Dianthins, DAPs, Dianthus
chinensis', Lychnin, Petroglaucin, Petrograndin, Saponaria
ocymoides RIP, Vacuolas saporin, Saporins, Vaccaria hispanica RIP,
Benincasins, Hispin, Byrodin's, Colocins, Cucumis figarei RIP,
Melonin, C. moschata RIP, Cucurmosin, Moschatins, Pepocin,
Gynostemmin, Gynostemma pentaphyllum RIP, Gypsophilin, Lagenin,
Luffaculin, Luffangulin, Luffin, MORs, Momordin II, Momorcharin's,
Momorcochin, Momorcochin-S, Sechiumin, Momorgrosvin, Trichoanguin,
Kirilowin, .alpha.-trichosanthin, TAP-29, Trichokirin,
Trichomislin, Trichosanthin, Karasurin, Trichomaglin, Trichobakin,
Crotin, Euserratin Antiviral Protein GAP-31, Gelonin, Hura
crepitans RIP, Curcin, Jathropa curcas RIP, Mapalmin, Manutins,
.alpha.-pisavin, Charibdin, Hyacinthus orientalis RIP, Musarmin,
Iris hollandica RIP, Cleroendrum aculeatum RIP, CIPs,), Crip-31,
Bouganin, Bougainvilla spectbilis RIP, Bougainvillea.times.buttiana
Antiviral protein 1 (BBAP1), Malic enzymes, MAP-S, pokeweed
antiviral proteins (PAP), PD-SI, DP-S2, Dodecandrin, PIP, PIP2,
Phytolacca octandra anti-viral proteins, Hordeum vulgare RIPs,
Hordeum vulgare sub sp. Vulgare Translational inhibitor II, Secale
cereale RIP, Tritin, Zea diploperemis RIPs, Malus.times.domestica
RIP, Momordica Anti-HIV Protein, Gelonium multiflorum, Mirabilis
expansa 1, phage MU1, betavulgin (Bvg), curcin 2, saporin 6, Maize
RIP (B-32), Tobacco RIP (TRIP), Beetins, Mirabilis antiviral
protein (MAP), Trichosanthin (TCS), luffins, Momorcharins,
Ocymoidin, Bryodin, Pepopsin, 13-trichosanthin, Camphorin, YLP,
Insularin, Barley RIP, Tritins, Lamjarin, Volvariella volvacea RIP
and the like of plant origin.
[0088] Polypeptide C may be selected from the group consisting of
Cyclotides, Siamycins, NP-06, Gramicidin A, Circulins, Kalatas,
Ginkbilobin, Alpha-Basrubin, Lunatusin, Sesquin, Tricyclon A,
Cycloviolacins, Polyphemusins, hfl-B5, Protegrins (Pig
Cathelicidin), Rat Defensins, Human .beta.-defensins, Temporins,
Caerins, Ranatuerins, Reptile Defensin, Piscidin's, Lactoferricin
B, Rabbit Neutrophils, Rabbit .alpha.-Defensin, Retrocyclins, Human
.alpha.-Defensins, Human .beta.-defensin III (HBD3), Rhesus
minidefensin (RTD-1,.theta.-defensin), rhesus .theta.-defensins,
Human neutrophil peptides, Cecropin As, Melittin, EP5-1, Magainin
2s, hybrid (CE-MA), hepcidin TH1-5, Epinecidin-1, Indolicidin,
Cathelicidin-4, LL-37 Cathelicidin, Dermaseptins, Maximins,
Brevinins, Ranatuerins, Esculentins, Maculatin 1.3, Maximin H5 and
Piscidins, Mundticin KS Enterocin CRL-35, Lunatusin, FK-13 (GI-20
is a derivative), Tachyplesins, Alpha-MSH, Antiviral protein Y3,
Palustrin-3AR, Ponericin L2, Spinigerin, Melectin, Clavanin B, Cow
cathelicidin's, Guinea pig cathelicidin CAP11, Sakacin 5X,
Plectasin, Fungal Defensin, GLK-19, lactoferrin (Lf) peptide 2,
Alloferon 1, Uperin 3.6, Dahlein 5.6, Ascaphin-8, Human Histatin 5,
Guineapig neutrophils, Mytilins, EP5-1,Hexapeptide (synthetic)
Corticostatin IV Rabbit Neutrophil 2, Aureins, Latarcin, Plectasin,
Cycloviolins, Vary Peptide E, Palicourein, VHL-1, Gaegurin 5,
Gaegurin 6 and the like (U.S. Pat. No. 8,076,284 B2; Kim, S. et al,
Peptides, 2003, 24, 945-953).
[0089] In particular, polypeptide C may be Gaegurin 5, Gaegurin 6,
their analogues, derivatives or fragments thereof, which may have
pro-apoptotic properties that may act upon drug sensitive and
multidrug resistant tumour cell lines.
[0090] A Cationic Antimicrobial Peptide (CAP) may be an
anti-microbial CAP that may have anticancer and/or antiviral
properties. CAPs may be a maximum of 100 amino acids in length.
CAPs may either be a naturally occurring CAP with sequence with
reported anticancer properties or a synthetic CAP sequence with
anticancer properties. CAPs may mostly be of animal origin.
However, there may also be CAPs, which are from plants, which
include but are not limited to cyclotides. For example, bacteria
CAPs may include but are not limited to Siamycin, NP-06 and
Gramicidin A. Plant CAPs may include Circulin A, B, Kalata B1 and
B8; Plant CAPs which may function as entry inhibitors may include
Kalata B8, Ginkbilobin, Alpha-Basrubin, Lunatusin and Sesquin,
Circulin A, C and D, Tricyclon A and Cycloviolacin H4. Animal CAPs
may include Polyphemusin I and II, hfl-B5, Protegrin (Pig
Cathelicidin), Rat Defensin NP1, NP2, NP3 and NP4, Human
.beta.-defensin I and II, Temporin A, Temporin-LTc, Temporin-Pta,
Caerin 1.1, Ranatuerin 6 and 9, Reptile Defensin and Piscidin 1 and
2, Lactoferricin B, Rabbit Neutrophil-1 Corticostatin III a, Rabbit
Neutrophil-3A, Rabbit .alpha.-Defensin, Retrocyclin-1,
Retrocyclin-2, Retrocyclin-3, Human .alpha.-Defensin HNP-1, 2,
3,4,5 & 6, Human .beta.-defensin III (HBD3), Rhesus
minidefensin (RTD-1,.theta.-defensin), RTD-2 rhesus
.theta.-defensin, RTD-3 rhesus .theta.-defensin, Human neutrophil
peptide-2, Human neutrophil peptide-3 and human neutrophil
peptide-4, Cecropin A, Melittin, EP5-1, Magainin 2, hepcidin TH1-5,
and Epinecidin-1, Indolicidin, Cathelicidin-4,
[0091] Human neutrophil peptide-1, LL-37 Cathelicidin,
Dermaseptin-S1, S4 and S9, Maximin 1, 3, 4 and 5, Brevinin 1,
Ranatuerin 2P, 6 and 9 Esculentin 2P, Esculentin-1 Arb, Caerin 1.1,
1.9 and 4.1, Brevinin-2-related, Maculatin 1.3, Maximin H5 and
Piscidin 1 and 2. Other CAPs may include Mundticin KS Enterocin
CRL-35, Lunatusin, FK-13 (GI-20 is a derivative), Tachyplesin I,
Alpha-MSH, Antiviral protein Y3, Piscidin 3, Palustrin-3AR,
Ponericin L2, Spinigerin, Melectin, Clavanin B, Cow cathelicidin
BMAP-27, BMAP-28, Guinea pig cathelicidin CAP11, Sakacin 5X,
Plectasin, Fungal Defensin, GLK-19, lactoferrin (Lf) peptide 2,
Kalata B8, Tricyclon A, Alloferon 1, Uperin 3.6, Dahlein 5.6,
Ascaphin-8, Human Histatin 5, Guineapig neutrophil CAP2 & CAP1,
Mytilin B & C, EP5-1, and Hexapeptide (synthetic) Corticostatin
IV Rabbit Neutrophil 2.
[0092] The Type 1 RIP may: [0093] (i) act as a pro-apoptotic
polypeptide which up regulate pro-apoptotic genes that may include
but not limited to caspase-12, Bax and the like, or down regulate
anti-apoptotic gene including but not limited to BcI-2 and the like
in tumour or cancer cells (Fan, J-M., et al, Mol Biotechnol, 2008,
39, 79-86); [0094] (ii) act as a DNA glycosylase/apurinic (AP)
lyase capable of irreversibly relaxing tumour or cancer cell
supercoiled DNA and catalyzing double-stranded breakage to form
inactive products; [0095] (iii) act in alternative cytochrome
pathways as well as Mn.sup.2+ and Zn.sup.2+ interactions with
negatively charged surfaces next to catalytic sites, facilitating
DNA substrate binding instead of directly participating in
catalysis (Wang et al, Cell, 1999, 99, 433-442); [0096] (iv) as an
RNA N-Glycosidase which hydrolyses the N-C glycosidic bond of
adenosine at position 4324 of the universally conserved
sarcin/ricin domain(S/R domain) of the 28S-rRNA in the eukaryotic
ribosome and render it incapable of carrying out protein synthesis
thus, functionally, blocking translation.
[0097] In particular, the Type 1 RIP may be selected from the group
consisting of .alpha.-Ebulitin, .beta.-Ebulitin, .gamma.-Ebulitin,
Nigritin f1, Nigritin f2, Amarandin-S, Amaranthus antiviral/RIP,
Amarandin-1, Amarandin-2, Amaranthin, Atriplex patens RIP, Beta
vulgaris RIP, .beta.-vulgin, Celosia cristata RIP, Chenopodium
album RIP, CAP30B, Spinacea oleracea RIP, Quinqueginsin, Asparin 1,
Asparin 2, Agrostin, Dianthin 29, DAP-30, DAP-32, Dianthin 30,
Dianthus chinensis RIP1, Dianthus chinensis RIP2, Dianthus
chinensis RIP3, Lychnin, Petroglaucin, Petrograndin, Saponaria
ocymoides RIP, Vacuolas saporin, Saporin-1, Saporin-2, Saporin-3,
Saporin-5, Saporin-6, Saporin-7, Saporin-9, Vaccaria hispanica RIP,
Benincasin, .alpha.-benincasin, .beta.-benincasin, Hispin, Byrodin
I, Byrodin II, Colocin I, Colocin 2, Cucumis figarei RIP, Melonin,
C. moschata RIP, Cucurmosin, Moschatin, Moschatin I, Moschatin II,
Moschatin III, Moschatin IV, Moschatin V, Pepocin, Gynostemmin I,
Gynostemmin II, Gynostemmin III, Gynostemmin IV, Gynostemmin V,
Gynostemma pentaphyllum RIP, Gypsophilin, Lagenin, Luffaculin,
Luffangulin, Luffin-alpha, Luffin-B, MOR-I, MOR-II, Momordin II,
Alpha-momorcharin, p-momorcharin, .gamma..delta.-momorcharin,
.gamma.-momorcharin, Momorcochin, Momorcochin-S, Sechiumin,
Momorgrosvin, Trichoanguin, .alpha.-kirilowin, .beta.-kirilowin,
.alpha.-trichosanthin, TAP-29, Trichokirin, Trichomislin,
Trichosanthin, Karasurin-A, Karasurin-B, Trichomaglin, Trichobakin,
Crotin 2, Crotin 3, Euserratin 1, Euserratin 2, Antiviral Protein
GAP-31, Gelonin, Hura crepitans RIP, Curcin, Jathropa curcas RIP,
Mapalmin, Manutin 1, Manutin 2, .alpha.-pisavin, Charibdin,
Hyacinthus orientalis RIP, Musarmin 1, Musarmin 2, Musarmin 3,
Musarmin 4, Iris hollandica RIP, Cleroendrum aculeatum RIP, CIP-29,
CIP-34, Crip-31, Bouganin, Bougainvilla spectbilis RIP,
Bougainvillea.times.buttiana Antiviral protein 1 (BBAP1), malic
enzyme 1 (ME1), ME2, MAP-S, pokeweed antiviral protein (PAPa-1),
PAPa-2, PAP-alpha, PAP-I, PAP-II, PAP-S, PD-SI, DP-S2, Dodecandrin,
Anti-viral protein PAP, PIP, PIP2, Phytolacca octandra anti-viral
protein, Phytolacca, octandra anti-viral protein II, Hordeum
vulgare RIP-I, Hordeum vulgare RIP-II, Hordeum vulgare sub sp.
Vulgare Translational inhibitor II, Secale cereale RIP, Tritin,
Zea, diploperemis RIP-I, Zea diploperemis RIP-II,
Malus.times.domestica RIP, Momordica Anti-HIV Protein (MAP30),
Gelonium multiflorum (GAP31), pokeweed antiviral protein (PAP),
Mirabilis expansa 1 (ME1), malic enzyme 2 (ME2),
Bougainvillea.times.buttiana antiviral protein 1 (BBAP1), phage
MU1, betavulgin (Bvg), curcin 2, saporin 6, Maize RIP (B-32),
Tobacco RIP (TRIP), beetin (BE), BE27, Mirabilis antiviral protein
(MAP), Trichosanthin (TCS), .alpha.-luffin, .alpha.-Momorcharin
(.alpha.-MMC), .beta.-MMC luffin, Ocymoidin, Bryodin, Pepopsin,
.beta.-trichosanthin, Camphorin, YLP, Insularin, Barley RIP,
Tritins, Lamjarin, Volvarielia volvacea RIP and the like of plant
origin.
[0098] In particular, polypeptide A may be a Retrocyclin,
polypeptide B may be MAP30 and polypeptide C may be a Dermaseptin.
More in particular, polypeptide A may be Retrocyclin 101 (RC101)
and polypeptide C may be Dermaseptin 1. A polypeptide comprising
RC101, MAP30 and Dermaseptin 1 as polypeptide A, B and C
respectively is termed RetroMAD1 in the present invention.
[0099] In particular, polypeptide A may comprise amino acid
sequence with SEQ ID NO: 4, a fragment or variant thereof,
polypeptide B may comprise amino acid sequence with SEQ ID NO:5, a
fragment or variant thereof, and polypeptide C may comprise amino
acid sequence with SEQ ID NO:6, a fragment or variant thereof.
[0100] More in particular, the fusion protein according to any
aspect of the present invention may comprise the amino acid
sequence SEQ ID NO:1. The fusion protein or the basic unit of the
fusion protein may have a molecular weight of about 30-50 kDa. In
particular, the molecular weight of the fusion protein may be 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 36.5, 37, 37.5,
37.8, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 kDa. The
fusion protein may comprise repeats of the basic unit. A skilled
person would understand that the weight of the fusion protein would
be dependent on the multiples of the basic unit present in the
protein. The nucleic acid coding for the fusion protein of SEQ ID
NO:1 may be found in SEQ ID NO:2. The sequences are provided in
Table 1b below.
[0101] In particular, polypeptide B may be Type 1 RIP, or a
fragment thereof, and polypeptide C may be Cationic Antimicrobial
Peptide or a fragment thereof; and--may be a direct linkage or a
linker peptide.
[0102] In one example, polypeptide A may be Avian .beta.-Defensin
103 (AVBD103), polypeptide B may be MAP30 and polypeptide C may be
Mytilin C10C. In another example, the fusion protein may comprise
the formula XIV:
C-B-C
TABLE-US-00002 TABLE 1b Sequences of polypeptides and
polynucleotides of the present invention. SEQ ID NO. Sequences 1
MKYLLPTAAAGLLLLAAQPAMAMGRICRCICGRGICRCICGVPGVGVPGVGGATGSDVNFDLSTATAKTY
TKFIEDFRATLPFSHKVYDIPLLYSTISDSRRFILLDLTSYAYETISVAIDVTNVYVVAYRTRDVSYFFK
ESPPEAYNILFKGTRKITLPYTGNYENLQTAAHKIRENIDLGLPALSSAITTLFYYNAQSAPSALLVLIQ
TTAEAARFKYIERHVAKYVATNFKPNLAIISLENQWSALSKQIFLAQNQGGKFRNPVDLIKPTGERFQVT
NVDSDVVKGNIKLLLNSRASTADENFITTMTLLGESVVEFPWALWKTMLKELGTMALHAGKAALGAAADT
ISQGTQVPGVGVPGVGKLAAALEHHHHHH 2
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccatgg
ggcgtatttgccgttgcatttgcggccgtggcatttgccgctgcatctgtggcgtgccgggtgttggtgt
tccgggtgtgggtggtgcgaccggatccgatgtgaactttgatctgagcaccgcgaccgcgaaaacctat
accaaattcatcgaagattttcgtgcgaccctgccgtttagccataaagtgtatgatatcccgctgctgt
atagcaccattagcgatagccgtcgttttattctgctggatctgaccagctatgcgtatgaaaccattag
cgtggcgattgatgtgaccaacgtgtatgtggtggcgtatcgtacccgtgatgtgagctactttttcaaa
gaaagcccgccggaagcgtacaacattctgtttaaaggcacccgtaaaattaccctgccgtataccggca
actatgaaaacctgcagaccgcggcgcataaaattcgtgaaaacatcgatctgggcctgccggccctgag
cagcgcgattaccaccctgttttattataacgcgcagagcgcgccgagcgcgctgctggtgctgattcag
accaccgcggaagcggcgcgttttaaatatattgaacgccacgtggcgaaatatgtggcgaccaacttta
aaccgaacctggccattattagcctggaaaaccagtggagcgccctgagcaaacaaatttttctggccca
gaaccagggcggcaaatttcgtaatccggtggatctgattaaaccgaccggcgaacgttttcaggtgacc
aatgtggatagcgatgtggtgaaaggcaacattaaactgctgctgaacagccgtgcgagcaccgcggatg
aaaactttattaccaccatgaccctgctgggcgaaagcgtggtggaattcccgtgggcgctgtggaaaac
catgctgaaagaactgggcaccatggcgctgcatgcgggtaaagcggcgctgggtgcggcagcggatacc
attagccagggcacccaggttccgggcgtgggcgttccgggcgttggtaagcttgcggccgcactcgagc
accaccaccaccaccactga 3 [VPXVG].sub.n 4 GRICRCICGRGICRCICG 5
GSDVNFDLSTATAKTYTKFIEDFRATLPFSHKVYDIPLLYSTISDSRRFILLDLTSYAYETISVAIDVTN
VYVVAYRTRDVSYFFKESPPEAYNILFKGTRKITLPYTGNYENLQTAAHKIRENIDLGLPALSSAITTLF
YYNAQSAPSALLVLIQTTAEAARFKYIERHVAKYVATNFKPNLAIISLENQWSALSKQIFLAQNQGGKFR
NPVDLIKPTGERFQVTNVDSDVVKGNIKLLLNSRASTADENFITTMTLLGESVVEFPW 6
ALWKTMLKELGTMALHAGKAALGAAADTISQGTQ
[0103] In another example, the fusion protein may be Amatilin,
RetroGAD1, Tamapal1 and the like. DNA and polypeptide sequences of
Amatilin, RetroGAD1, and Tamapal1 are presented in Tables 1d and
1e.
[0104] Investigations on thermal behaviour of drug samples are
important for obtaining information for their processing in
pharmaceutical industry, for predicting their shelf lives and also
for suitable storage conditions. These drugs were shown to be
thermally stable.
TABLE-US-00003 TABLE 1c The polypeptides used for each drug.
Example Polypeptide A Polypeptide B Polypeptide C Fusion peptide
Defensin RIP CAP RetroMAD1 Retrocyclin 101 MAP30 Dermaseptin1
RetroGAD1 Retrocyclin 101 GAP31 Dermaseptin1 Tamapal1 Tachyplesin
MAP30 Alloferon1 Amatilin AVBD103 MAP30 Mytillin C10C
TABLE-US-00004 TABLE 1d DNA sequences of Amatilin, RetroGAD1 and
Tamapal1 SEQ Fusion ID Protein NO. DNA Sequence Amatilin 37
GGGCAGTGAGCGGAAGGCCCATGAGGCCAGTTAATTAAGAGGTACCGAATTCTCAT
TCGGTTTGTGTAGATTGAGAAGAGGTTTCTGTGCTCACGGTAGATGTAGATTCCCA
TCCATCCCAATCGGTAGATGTTCCAGATTCGTTCAGTGTTGTAGAAGAGTTTGGGT
CCCAGGTGTTGGTGTTCCAGGTGTTGGAGGTGCTACTGGTTCTGATGTTAACTTCG
ACTTGTCCACTGCTACTGCTAAGACTTACACTAAGTTCATCGAGGACTTCAGAGCT
ACTTTGCCATTCTCCCACAAGGTTTACGACATCCCTTTGTTGTACTCCACTATCTC
CGACTCCAGAAGATTCATCTTGTTGAACTTGACTTCCTACGCTTACGAGACTATCT
CCGTTGCTATCGACGTTACAAACGTTTACGTTGTTGCTTACAGAACTAGAGATGTT
TCCTACTTCTTCAAAGAGTCCCCACCAGAGGCTTACAACATCTTGTTCAAGGGTAC
TAGAAAGATCACTTTGCCATACACTGGTAACTACGAGAACTTGCAGACTGCTGCTC
ACAAGATCAGAGAGAACATCGACTTGGGTTTGCCAGCTTTGTCCTCCGCTATCACT
ACTTTGTTCTACTACAACGCTCAGTCCGCTCCATCCGCTTTGTTGGTTTTGATCCA
GACTACTGCTGAGGCTGCTAGATTCAAGTACATCGAGAGACACGTTGCTAAGTACG
TTGCTACAAACTTCAAGCCAAACTTGGCTATCATCTCCTTGGAGAACCAGTGGTCT
GCTTTGTCCAAGCAGATCTTCTTGGCTCAAAACCAGGGTGGTAAGTTCAGAAACCC
AGTCGACTTGATCAAGCCAACCGGTGAGAGATTCCAGGTTACTAATGTTGACTCCG
ACGTTGTTAAGGGTAACATCAAGTTGTTGTTGAACTCCAGAGCTTCCACTGCTGAC
GAGAACTTCATCACTACTATGACTTTGTTGGGTGAGTCCGTTGTTAACTCCTGTGC
TTCCAGATGTAAGGGTCACTGTAGAGCTAGAAGATGTGGTTACTACGTTTCCGTTC
TGTACAGAGGTAGATGTTACTGTAAATGTTTGAGATGTGTCCCCGGTGTTGGAGTC
CCTGGTGTCGGTGCGGCCGCGAGCTCATGGCGCGCCTAGGCCTTGACGGCCTTCCG CCAATTCGC
RetroGAD1 38
CGAATTGGCGGAAGGCCGTCAAGGCCACGTGTCTTGTCCAGGTACCGAATTCGGAA
TCTGTAGATGCATCTGCGGTAGAGGTATCTGCAGATGTATTTGTGGAAGAGTCCCA
GGTGTTGGTGTTCCAGGTGTTGGAGGTGCTACTGGTTCTGGTTTGGACACTGTTTC
ATTCTCCACTAAGGGTGCTACTTACATCACTTACGTTAACTTTTTGAACGAGTTGA
GAGTTAAGTTGAAGCCAGAGGGTAACTCCCACGGTATCCCTTTGTTGAGAAAGAAG
TGTGACGACCCAGGTAAGTGTTTCGTTTTGGTTGCTTTGTCCAACGACAACGGTCA
GTTGGCTGAGATTGCTATCGACGTTACTTCCGTTTACGTTGTTGGTTACCAGGTTA
GAAACAGATCCTACTTCTTCAAGGACGCTCCAGACGCTGCTTACGAAGGTTTGTTC
AAGAACACTATCAAGACTAGATTGCACTTCGGTGGTTCCTACCCATCTTTGGAAGG
TGAGAAGGCTTACAGAGAGACTACTGACTTGGGTATCGAGCCATTGAGAATCGGTA
TCAAGAAGTTGGACGAGAACGCTATCGACAACTACAAGCCAACTGAGATCGCTTCC
TCCTTGTTGGTTGTTATCCAGATGGTTTCCGAGGCTGCTAGATTCACTTTCATCGA
GAACCAGATCAGAAACAACTTCCAGCAGAGAATCAGACCAGCTAACAACACTATTT
CCTTGGAGAACAAGTGGGGTAAGTTGTCCTTCCAGATCAGAACATCCGGTGCTAAC
GGTATGTTCTCTGAGGCTGTTGAGTTGGAGAGAGCTAACGGTAAGAAGTACTACGT
TACTGCTGTTGACCAGGTTAAGCCAAAGATCGCTTTGTTGAAGTTCGTTGACAAGG
ACCCAAAGGGTTTGTGGTCCAAGATCAAAGAGGCTGCTAAGGCTGCTGGTAAGGCT
GCTTTGAATGCTGTTACTGGTTTGGTTAACCAGGGTGACCAACCATCTGTCCCTGG
TGTTGGAGTCCCTGGTGTCGGTGCGGCCGCGAGCTCTGGAGCACAAGACTGGCCTC
ATGGGCCTTCCGCTCACTGC Tamapal1 39
GGATCCGTTCCGGGTGTGGGTGTTCCGGGTGTTGGTAAATGGTGTTTCGTGTTTGT
TATCGCGGTATTTGTTATCGTCGTTGTCGTGTGCCAGGCGTTGGCGTTCCAGGCGT
GGGTGGTGCAACCGGTAGTGATGTTAATTTTGATCTGAGCACCGCAACCGCAAAAA
CCTATACCAAATTTATCGAAGATTTTCGTGCAACCCTGCCGTTTAGCCATAAAGTT
TATGATATTCCGCTGCTGTATAGCACCATTAGCGATAGCCGTCGTTTTATTCTGCT
GAATCTGACCAGCTATGCCTATGAAACCATTAGCGTTGCAATTGATGTGACCAATG
TTTATGTTGTTGCATATCGTACCCGTGATGTGAGCTATTTTTTCAAAGAAAGCCCT
CCGGAAGCCTATAACATTCTGTTTAAAGGCACCCGCAAAATCACCCTGCCGTATAC
CGGTAATTATGAAAATCTGCAGACCGCAGCACATAAAATTCGCGAAAATATTGATC
TGGGTCTGCCTGCACTGAGCAGCGCAATTACCACCCTGTTTTATTACAATGCACAG
AGCGCACCGAGCGCACTGCTGGTTCTGATTCAGACCACCGCAGAAGCAGCACGCTT
TAAATACATTGAACGTCATGTTGCCAAATACGTGGCCACCAACTTTAAACCGAATC
TGGCAATTATTAGCCTGGAAAATCAGTGGTCAGCACTGAGCAAACAAATTTTTCTG
GCACAGAATCAGGGTGGCAAATTTCGTAATCCGGTTGATCTGATTAAACCG
ACCGGTGAACGTTTTCAGGTTACCAATGTTGATAGTGATGTGGTGAAAGGCAACAT
TAAACTGCTGCTGAATAGCCGTGCAAGCACCGCAGATGAAAACTTTATTACCACCA
TGACCCTGCTGGGTGAAAGCGTTGTTAATGTTCCTGGTGTTGGCGTGCCTGGTGTT
GGTCATGGTGTTAGCGGTCATGGTCAGCATGGTGTTCATGGTTAAAAGCTT
TABLE-US-00005 TABLE 1e Polypeptide sequences of Amatilin,
RetroGAD1 and Tamapal1 SEQ Fusion ID Protein NO. Protein Sequence
Amatilin 28
SFGLCRLRRGFCAHGRCRFPSIPIGRCSRFVQCCRRVWVPGVGVPGVGGATGSDVNF
DLSTATAKTYTKFIEDFRATLPFSHKVYDIPLLYSTISDSRRFILLNLTSYAYETIS
VAIDVTNVYVVAYRTRDVSYFFKESPPEAYNILFKGTRKITLPYTGNYENLQTAAHK
IRENIDLGLPALSSAITTLFYYNAQSAPSALLVLIQTTAEAARFKYIERHVAKYVAT
NFKPNLAIISLENQWSALSKQIFLAQNQGGKFRNPVDLIKPTGERFQVTNVDSDVVK
GNIKLLLNSRASTADENFITTMTLLGESVVNSCASRCKGHCRARRCGYYVSVLYRGR
CYCKCLRCVPGVGVPGVG RetroGAD 36
GICRCIGRGICRCICGRVPGVGVPGVGGATGSGLDTVSFSTKGATYITYVNFLNELR 1
VKLKPEGNSHGIPLLRKKCDDPGKCFVLVALSNDNGQLAEIAIDVTSVYVVGYQVRN
RSYFFKDAPDAAYEGLFKNTIKTRLHFGGSYPSLEGEKAYRETTDLGIEPLRIGIKK
LDENAIDNYKPTEIASSLLVVIQMVSEAARFTFIENQIRNNFQQRIRPANNTISLEN
KWGKLSFQIRTSGANGMFSEAVELERANGKKYYVTAVDQVKPKIALLKFVDKDPKGL
WSKIKEAAKAAGKAALNAVTGLVNQGDQPSVPGVGVPGVG Tamapal1 34
VPGVGVPGVGKWCFRVCYRGICYRRCRVPGVGVPGVGGATGSDVNFDLSTATAKTYT
KFIEDFRATLPFSHKVYDIPLLYSTISDSRRFILLNLTSYAYETISVAIDVTNVYVV
AYRTRDVSYFFKESPPEAYNILFKGTRKITLPYTGNYENLQTAAHKIRENIDLGLPA
LSSAITTLFYYNAQSAPSALLVLIQTTAEAARFKYIERHVAKYVATNFKPNLAIISL
ENQWSALSKQIFLAQNQGGKFRNPVDLIKPTGERFQVTNVDSDVVKGNIKLLLNSRA
STADENFITTMTLLGESVVNVPGVGVPGVGHGVSGHGQHGVHG
[0105] Modifications and changes may be made in the structure of
the peptides of the present invention and DNA segments, which
encode them and still obtain a functional molecule that encodes a
protein or peptide with desirable characteristics. The amino acids
changes may be achieved by changing the codons of the DNA sequence.
For example, certain amino acids may be substituted for other amino
acids in a protein structure without appreciable loss of
interactive binding capacity with structures such as, for example,
tumour or cancer cell-binding regions of fusion proteins. Since it
is the interactive capacity and nature of a protein that defines
that protein's biological functional activity, certain amino acid
sequence substitutions can be made in a protein sequence, and, of
course, its underlying DNA coding sequence, and nevertheless obtain
a protein with like properties. Various changes may be made in the
peptide sequences of the disclosed compositions, or corresponding
DNA sequences, which encode said proteins without appreciable loss
of their biological utility or activity. Amino acid substitutions
of the fusion protein according to the present invention may be
possible without affecting the antitumour or anticancer effect of
the isolated peptides of the invention, provided that the
substitutions provide amino acids having sufficiently similar
properties to the ones in the original sequences.
[0106] The fusion peptide according to any aspect of the present
invention may be thermostable over a prolonged period of time.
Thermostability is an industrially significant attribute as
cold-chain transportation will greatly increase logistics and
handling costs that will contribute to the overall total cost of
the medication. Also, if the drug is to be carried about to be
consumed before meals, patient compliance will suffer if the
requirement of low temperature storage in an absolute necessity.
Thus, the ability to remain stable for 7 days even at elevated
temperatures will allow for a wider usage and application of the
therapeutic protein.
[0107] The fusion protein may further comprise a pharmaceutically
acceptable carrier, excipient, adjuvant, diluent and/or detergent.
Such formulations therefore include, in addition to the fusion
protein, a physiologically acceptable carrier or diluent, possibly
in admixture with one or more other agents such as other antibodies
or drugs, such as an antibiotic. Suitable carriers include, but are
not limited to, physiological saline, phosphate buffered saline,
phosphate buffered saline glucose and buffered saline.
Alternatively, the fusion protein may be lyophilized (freeze dried)
and reconstituted for use when needed by the addition of an aqueous
buffered solution as described above. Routes of administration are
routinely parenteral, including intravenous, intramuscular,
subcutaneous and intraperitoneal injection or oral delivery. The
administration can be systemic and/or local. The medicament may be
used for topical or parenteral administration, such as
subcutaneous, intradermal, intraperitoneal, intravenous,
intramuscular or oral administration. For this, the fusion protein
may be dissolved or suspended in a pharmaceutically acceptable,
preferably aqueous carrier. The medicament may contain excipients,
such as buffers, binding agents, blasting agents, diluents,
flavours, lubricants, etc.
[0108] In particular, the fusion protein may be produced as a solid
dose by means of Supercritical Fluid Drying (SCFD) used to dry and
produce a micronized form of powdered free-flowing RetroMAD1. The
powder may be for incorporation into tablets, capsules and animal
feed pellets whether for terrestrial or aquatic application. This
allows for high process yields and may enable further ease of oral
drug delivery in tablet and/or capsule form.
[0109] In particular, the fusion protein may be administered
orally. In particular, the presence of MAP30 surprisingly renders
the fusion protein according to any aspect of the present invention
stable for oral administration. In particular, the fusion protein
may be administered with or before food. More in particular, when
the fusion protein is administered before food, it may be done with
a drink for example water. In the case of aquatic animals that do
not `drink`, it may be effectively administered by top-coating the
feed pellets with the fusion protein. Furthermore, they can be
coated further with proteins to prevent leaching. A non-limiting
example is the use of proteins from chicken eggs and the like to
protect against leaching.
[0110] The fusion protein according to any aspect of the present
invention may be capable of maintaining its form in the digestive
tract without fragmentation or enzymatic digestion. In one example,
the fusion protein may be in a liquid form. In particular, the
fusion protein may be ingested, as a drink diluted with water, or
the like, and the retention time in either stomach or duodenum is
only a matter of minutes allowing the protein to reach its target
point without being digested.
[0111] The dosage of the fusion protein according to the present
invention to be administered to a non-human animal may vary with
the precise nature of the condition being treated and the recipient
of the treatment. The dose will generally be in the range of about
0.005 to about 1000 mg for an adult patient, usually administered
daily for a period between 1 day to 2 years. In particular, the
daily dose may be 0.5 to 100 mg per day. In particular the daily
dose may be about 0.8, 1, 1.2, 1.5, 2, 2.5, 3.2, 4, 4.5, 5, 10, 15,
20, 30, 45, 50, 75, 80, 90, 95 mg per day. The dosage may be
applied in such a manner that the ligand may be present in the
medicament in concentrations that provide in vivo concentrations of
said ligand in a patient to be treated of between 0.001 mg/kg/day
and 5 mg/kg/day. In one embodiment, the medicament, the peptide or
ligand according to the invention is present in an amount to
achieve a concentration in vivo of 1 .mu.g/ml or above with a
maximum concentration of 100 .mu.g/ml. the dosage regime may be
varied depending on the results on the patient.
[0112] The fusion protein may be pegylated to aid in the medicament
being suitable for oral delivery. In particular, the fusion protein
may be pegylated with any PEG known in the art. The PEG may be
selected from the group consisting of but not limited to PEG200,
300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,
1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300,2400, 2500,
2600, 2700, 2800, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500,
4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000 and the like.
[0113] SPF animal may be free from only the pathogens that they
have been tested for. In shrimp for example, typically this may
consist of the viral pathogens which are known to cause major
losses to the shrimp culture industry, including WSSV, YHV, TSV,
IHHNV, BPV, HPV 3 and the like. However, when new diseases may
emerge from mutations of previously non-pathogenic organisms--i.e.
the highly mutable RNA viruses the SPF animal may not survive.
Hence, it remains a possibility that importation of SPF shrimp may
not rule out simultaneous importation of pathogens. Also, if SPF
shrimp are stocked into facilities with high viral loads,
substantial mortality can result as they are not necessarily more
resistant to these diseases than non-SPF shrimp, and in some cases,
less so. They may thus be more suited to culture in biosecure
systems, which may explain the reliance of the big, non-biosecure
pond farms of Latin America on SPR (Specific Pathogen Resistant),
rather than SPF shrimp. Accordingly, even though SPF animals have
their advantages, they have their limitations and an SPR animal may
be needed that may be capable of resistance to all pathogens.
[0114] In another aspect of the present invention, there is
provided a method of producing at least one specific pathogen
resistant (SPR) non-human animal, the method comprising: [0115] (a)
producing a specific pathogen free animal according to any method
of the present invention; and [0116] (b) selective breeding of a
male and female SPF non-human animal to produce a SPR non-human
animal offspring.
[0117] According to a further aspect of the present invention,
there is provided a specific pathogen free or resistant non-human
animal produced by any method of the present invention. The animal
may be an aquatic animal. More in particular, the aquatic animal
may be a prawn of any species or a fish. In one example, the animal
may be a non-aquatic animal for example a bird like a chicken and
the like. The animal may be of any age and include every stage of
the life-cycle of the animal. In particular, the animal may include
an egg, larvae and the like of the animal.
[0118] A person skilled in the art will appreciate that the present
invention may be practised without undue experimentation according
to the method given herein. The methods, techniques and chemicals
are as described in the references given or from protocols in
standard biotechnology and molecular biology text books.
[0119] The fusion protein and/or pharmaceutical composition
according to any aspect of the present invention may result in no
or substantially no toxic side effects when taken by the
animal.
[0120] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples which are provided by way of illustration, and are not
intended to be limiting of the present invention.
EXAMPLES
[0121] Standard molecular biology techniques known in the art and
not specifically described were generally followed as described in
Sambrook and Green, Molecular Cloning: A Laboratory Manual, Cold
Springs Harbor Laboratory (Fourth Edition), New York (2012).
Example 1
Construction and Design of Expression Vector
[0122] The gene encoding RetroMAD1 A-B-C with SEQ ID NO:1 was
synthesized and cloned into backbone of vector pGA4 at the
KpnI/SacI site by contract service (GeneArt AG, Germany). The
expected product size was 1140 bp, which encoded a 379 amino acid
and an expected size of 41.2 kDa. The polynucleotide sequence and
the translated polypeptide sequence are shown in FIG. 1 from PCT.
The gene was sub-cloned into a pET expression vector (Novagen),
pET-26(b) at the NcoI/HindIII sites. Kanamycin was used as a marker
for selection and maintenance of culture purposes. This vector was
inducible under the addition of
isopropyl-beta-D-thiogalactopyranoside (IPTG). The plasmid, pRMD1
was then transformed into BL21(DE23) cells (Novagen) and plated on
a selective media with Kanamycin.
Expression of RetroMAD1 from E. coli
[0123] One recombinant clone was grown in 10 ml of LB Bertani
(DIFCO) medium, supplemented with 30 .mu.g/ml kanamycin, at
37.degree. C. overnight. This culture was used to inoculate 100 ml
of LB Bertani supplemented with 30 .mu.g/ml kanamycin and grown at
37.degree. C. until the optical reading was 0.4-0.6 at 600 nm. IPTG
was added at 1.0 mM final concentration. The growth period
continued for 3 hours. An SDS-PAGE analysis of the fraction of
RetroMAD1 in cells extracted in electrophoresis loading buffer
showed that a protein had a molecular mass of about 37.5 kDa, the
expected molecular size of RetroMAD1 was produced in the induced
cells only (FIG. 2A). Further solubility analysis by SDS-PAGE
revealed that RetroMAD1 was found in the pellet fraction and not in
the supernatant fraction of the E. coli indicating that the protein
was expressed and produced as inclusion bodies as shown in FIG.
2B.
Isolation and Purification of RetroMAD1
[0124] Cells from 100 ml of induced culture were harvested by
centrifugation for 10 min at 5000.times.g at 15.degree. C. The
cells were suspended in a lysis buffer containing 20 mM Tris-HCl
(pH 7.5), 10 mM EDTA and 1% Triton-X 100. Cells were disrupted by
sonication. The insoluble fraction was isolated from the soluble
fraction by centrifugation at 8,000.times.g for 20 min. The
supernatant was discarded and the pellet was further washed by
repeating the same step. The pellet was further washed twice with
RO water by resuspension via sonication and separation by
centrifugation.
Solubilization of RetroMAD1
[0125] The insoluble material was dissolved and sonicated in 10 ml
of 5-8 Urea or 6M Guanidine Hydrochloride and supplemented with
2-5% of Sodium-lauryl sarcosine and 100 mM .beta.-mercaptoethanol.
The solubilisation was carried out overnight. The solubilised
protein was separated from the bacterial cell wall by
centrifugation at 8,000.times.g for 20 minutes.
Refolding of RetroMAD1
[0126] Renaturation of the protein was carried out by using
dialysis. The protein (10 ml) was dialysed in a 15 kDa molecular
weight cut-off dialysis membrane (Spectra/Por Lab). The protein was
dialysed in 5 L of RO water with the pH of 11.0 adjusted by NaOH.
Incubation was done at room temperature for 15-20 hours. The
refolded protein was transferred to a 50 ml tube and centrifuged at
8,000.times.g to separate any insoluble material. Renatured protein
was stored at -20.degree. C. until further use. The bioactivity of
RetroMAD1 in the following examples is proof of successful
refolding of the protein.
Example 2
Elimination of Hepatopancreatic ParvoVirus (HPV) from Shrimp
Shrimp Culture and RetroMAD1 Treatment
[0127] Naturally infected HPV shrimp (150 pieces) was obtained from
a local aquarium shop. Twenty pieces of of randomly selected shrimp
was selected for DNA extraction to confirm for HPV
(Hepatopancreatic Parvo Virus) infection in the population. For the
experiment, 56 shrimps were reared in two 20 liters tank (24 each)
containing de-chlorinated fresh water equipped with aeration. Water
exchange was carried out at 20% every two days. Shrimps were
acclimatized for one week before the experiment.
[0128] For the experiment both tanks were given 0.25 mg of feed
daily, divided into 3 meals. Treated tanks were a given a dose of
25 ug of RetroMAD1 absorbed into the commercial feed for each meal
for four days while the control was given sterile water absorbed
into the feed. After the end of the experiment (day 4), 24 pieces
of shrimp were still alive in the treated tank while 23 pieces were
still alive. All shrimp were subjected to a whole-body DNA
extraction.
DNA Extraction
[0129] DNA was extracted from whole body using salting-procedure
(Aljanabi, S. M. and L. Martinez, 1997. Universal and rapid
salt-extraction of high quality genomic DNA for PCR-based
techniques. Nucleic Acid Res., 25: 4692-4693). Primers used in this
experiment was HPVF: 5'-ACA-CTC-AGC-CTC-TAC-CTT-GT 3' and HPVR: 5'
-GCA-TTA-CAA-GAG-CCA-AGC-AG-3'. Thirty-five cycles of amplification
were performed at 30 s at 94.degree. C., 30 s at 55.degree. C., and
50 s at 72.degree. C. for both primer pairs. The expected PCR
products were analyzed in a 2% agarose gel, with the expected band
of 441 by as shown in FIG. 3.
Results
[0130] PCR analysis showed that RetroMAD1 in the treated Paleonetes
sp tank, 92% (22/24) were HPV negative while 8% (2/24) were HPV
positive. In the control non-treated group, 95% (22/23) were HPV
positive while 5 percent (1/23) were HPV negative.
Example 3
Effect of RetroMAD1 on WSSV-Infected Shrimp
Shrimp Culture
[0131] White Leg Shrimp Penaeus vannamei (36 pieces) at an average
of 8.0.+-.0.5 grams were used in this experiment they were obtained
from pond-reared from SPF (specific pathogen free) post-larvae
obtained from commercial hatcheries. Treated sea water was obtained
from the hatchery. Cultures of healthy shrimp were performed in a
recirculation system (equipped with filter and aeration) with a
salinity of 28-32 ppt in a bio-secure laboratory at 28.degree. C.
They were acclimatized 1 week before the infection experiment. Two
groups of 18 prawns were reared in a 90 liter tank with and
individual filter (FIG. 4).
WSSV Infection
[0132] Prawns were orally challenged by feeding frozen flesh from
WSSV-PCR positive prawns obtained from a recently WSSV-killed pond
at approximately 5% of body weight on the first day. The next day,
were given RetroMAD1 at a concentration of 0.1 mg/g body weight by
coating it into a commercial feed. They were given the medicated
feed for all meals (4 times a day). Observation was carried in term
mortality after 24 hours of infection. At the end of the
experiments, all live prawns were collected. These moribund and
live prawns were subjected to PCR analysis.
DNA Extraction
[0133] DNA was extracted from the pleopod using salting-procedure
(Aljanabi, S. M. and L. Martinez, 1997). Primers used in this
experiment was WSVF: 5'-TAT-TGT-CTC-TCC-TGA-CGT-AC-3' and WSVR: 5'
-CAC-ATT-CTT-CAC-GAG-TCT-AC-3'. Thirty-five cycles of amplification
were performed at 30 s at 94.degree. C., 30 s at 55.degree. C., and
50 s at 72.degree. C. for both primer pairs. The expected PCR
products were analyzed in a 2% agarose gel, with the expected band
of 298 bp.
Result
[0134] In the control tank, mortalities began on day 3
post-challenge and by day 8, nearly all of the 18 prawns were dead.
By day 9 post-challenge, 100% mortality was observed in the control
tank showing that the WSSV-infected carcass used was very much
capable of causing 100% mortality within 9 days post-oral
infection. In the treated tank, no mortality was observed until day
fourteen.
[0135] PCR analysis showed that all moribund prawns (18/18) from
the control group had high level of WSSV. Interestingly, in the
treated group 9/18 had low infection, 3/18 had very low infection
while 6/18 undetected (FIG. 5).
Example 4
Time Needed for Sero-Reversal to Occur in MBV-Infected Shrimp
[0136] Monodon Baculovirus (MBV) is an OIE `listed for
notification` shrimp DNA virus that has historically contributed to
significant commercial losses in shrimp farming. A total of 5 MBV
highly-PCR positive 6 g Penaeus vannamei were detected from a
subsample of 20 shrimp obtained live from a commercial shrimp farm
in Tawau, Malaysia and tested based on sacrificing one pleopod for
DNA extraction. These were individually kept in separate 10 L
aerated plastic aquariums that had 30% daily water exchange at 30
ppt salinity for a 1 week acclimation period. Ammonia and Nitrite
were monitored to ensure adequate water quality. They were then fed
a commercial pellet feed (Charoen Pokphand) once a day with 100 mg
each feeding. The intentionally low feeding rate was to ensure that
all the feed would be consumed and not contribute to developing
ammonia in the experimental tank. RetroMAD1 at 2 mg/ml
concentration was added at 150 ml/kg to prepare the stock feed for
the experiment by applying it on the surface of the feed followed
by convection drying at 35.degree. C. in an oven. These were then
stored at 4.degree. C. in a refrigerator for the duration of the
experiment. After a week, another pleopod was surgically removed
and tested with standard Polymerase Chain Reaction (PCR) against
the highly conserved coat protein of the virus. The primers used
were: MBV F: 5' TACCATAAGCTAGCATACGCC 3' and MBV R: 5'
GGGGGCACAAGTCTCACAAG 3'. Nucleic acid isolation and the PCR
protocol used were the same as Example 3 above. The size of the PCR
product was 305 bp.
[0137] At the end of week 1, it was found that all the resultant
pleopod samples were still PCR positive but at the end of week 2,
all the samples from another surgically removed pleopod showed that
they had all become PCR negative. It is therefore suggested that
sero-reversal in MBV infected shrimp from PCR positive to PCR
negative takes approximately 2 weeks after feeding with
RetroMAD1.
TABLE-US-00006 TABLE 2 Results of PCR for MBV post RetroMAD1
treatment PCR Result P. vannamei Day 0 Day 7 Day 14 Animal 1
Positive Positive Negative Animal 2 Positive Positive Negative
Animal 3 Positive Positive Negative Animal 4 Positive Positive
Negative
Example 5
Stability of RetroMAD1 to Trypsin at pH8
[0138] The ability of RetroMAD1 to withstand action of digestive
enzymes acting at their pH optima is shown in Table 3 below.
[0139] 50 mM DTT was prepared and added into pre-dissolved
RetroMAD1 protein (1:1) made according to Example 1 and mixed. This
was heated at 95.degree. C. for 10 minutes and used to carry out
enzyme assays with proteases such as Trypsin (pH8) (Lonza,
Walkersville), .alpha.-Chymotrypsin (pH8) (Sigma-Aldrich) and
Pepsin (pH2) (Sigma-Aldrich). After 10 minutes of heating at
95.degree. C., the reaction was allowed to cool to room temperature
(Approx. 10 mins) and proteases added to a final ratio of 1:100
(w/w) (protease:protein). This was incubated at 37.degree. C. for 2
hours and protease activity terminated by incubating the mixture at
65.degree. C. for 15 minutes. SDS-PAGE was used to analyze the
fragments.
[0140] Other fusion proteins provided in Table 4 were made
according to the method of Example 1 and the results of their
fragmentation provided in Table 3.
TABLE-US-00007 TABLE 3 Results of fragmentation of fusion proteins
according to the present invention No of bands after proteas
digestion Size of SEQ ID Chymotr Drug drug NO: Structure of drug
Pepsin Trypsin psin AM 40 kDa 28 A-B-C No No No
(AVBD103-MAP30-MYTILINC10C) fragment fragment fragment CT 36 kDa 29
A-A-B-C No No No (CERCROPIN A-CERCROPIN D- fragment fragment
fragment TAP29-DAP30-LATARCIN 2A) AB 32 kDa 30 (RETROCYCLIN
101-MORMODICA No No No ANTI-HIV PROTEIN 30) fragment fragment
fragment BA 32 kDa 31 (MORMODICA ANTI-HIV PROTEIN No No No 30-
RETROCYCLIN 101) fragment fragment fragment BC 35 kDa 32 (MORMODICA
ANTI-HIV PROTEIN No No No 30- DERMASEPTIN 1) fragment fragment
fragment CB 35 kDa 33 DERMASEPTIN 1- MORMODICA No No No ANTI-HIV
PROTEIN 30 fragment fragment fragment Tamapal1 35.93 kDa 34 C-B-C
No No No TACHYPLESIN- MAP30- fragment fragment fragment ALLOFERON1
K5 36.55 kDa 35 C-B-D No No No (GAEGURIN 5-MAP30-(KLAKLAK)2
fragment fragment fragment RetroMAD1 41.2 kDa 1 A-B-C No No No
(RETROCYCLIN 101- MAP30- fragment fragment fragment DERMASEPTIN 1)
RetroGAD1 35.29 kDa 36 A-B-C No No No (RETROCYCLIN 101- GAP31-
fragment fragment fragment DERMASEPTIN 1) indicates data missing or
illegible when filed
TABLE-US-00008 TABLE 4 Examples of fusion proteins according to the
present invention SEQ ID NO: SEQUENCE 27 [G-G-G-S].sub.n 28
SFGLCRLRRGFCAHGRCRFPSIPIGRCSRFVQCCRRVWVPGVGVPGVGGATGSDVNFDLSTATAKTYTK
FIEDFRATLPFSHKVYDIPLLYSTISDSRRFILLNLTSYAYETISVAIDVTNVYVVAYRTRDVSYFFKE
SPPEAYNILFKGTRKITLPYTGNYENLQTAAHKIRENIDLGLPALSSAITTLFYYNAQSAPSALLVLIQ
TTAEAARFKYIERHVAKYVATNFKPNLAIISLENQWSALSKQIFLAQNQGGKFRNPVDLIKPTGERFQV
TNVDSDVVKGNIKLLLNSRASTADENFITTMTLLGESVVNSCASRCKGHCRARRCGYYVSVLYRGRCYC
KCLRCVPGVGVPGVG 29
LEKRKWKLFKKIEKVGQRVRDAVISAGPAVATVAQATALAKNVPGVGVPGVGGATGSDVSFRLSGATSK
KKVYFISNLRKALPNEKKLYDIPLVRSSSGSKATAYTLNLANPSASQYSSFLDQIRNNVRDTSLIYGGT
DVAVIGAPSTTDKFLRLNFQGPRGTVSLGLRRENLYVVAYLAMDNANVNRAYYFKNQITSAELTALFPE
VVVANQKQLEYGEDYQATEKNAKITTGDQSRKELGLGINLLITMIDGVNKKVRVVKDEARFLLIAIQMT
AEAARFRYIQNLVTKNFPNKFDSENKVIQFQVSWSKISTAIFGDCKNGVFNKDYDFGFGKVRQAKDLQM
GLLKYLGRPKSSSIEANSTDDTADVLVPGVGVPGVG KTCENLADTFRGPCFATSNC 30
MGRICRCICGRGICRCICGVPGVGVPGVGGSDVNFDLSTATAKTYTKFIEDFRATLPFSHKVYDIPLLY
STISDSRRFILLDLTSYAYETISVAIDVTNVYVVAYRTRDVSYFFKESPPEAYNILFKGTRKITLPYTG
NYENLQTAAHKIRENIDLGLPALSSAITTLFYYNAQSAPSALLVLIQTTAEAARFKYIERHVAKYVATN
FKPNLAIISLENQWSALSKQIFLAQNQGGKFRNPVDLIKPTGERFQVTNVDSDVVKGNIKLLLNSRAST
ADENFITTMTLLGESVVEFPW 31
MGSDVNFDLSTATAKTYTKFIEDFRATLPFSHKVYDIPLLYSTISDSRRFILLDLTSYAYETISVAIDV
TNVYVVAYRTRDVSYFFKESPPEAYNILFKGTRKITLPYTGNYENLQTAAHKIRENIDLGLPALSSAIT
TLFYYNAQSAPSALLVLIQTTAEAARFKYIERHVAKYVATNFKPNLAIISLENQWSALSKQIFLAQNQG
GKFRNPVDLIKPTGERFQVTNVDSDVVKGNIKLLLNSRASTADENFITTMTLLGESVVEFPWVPGVGVP
GVGGRICRCICGRGICRCICG 32
MGSDVNFDLSTATAKTYTKFIEDFRATLPFSHKVYDIPLLYSTISDSRRFILLDLTSYAYETISVAIDV
TNVYVVAYRTRDVSYFFKESPPEAYNILFKGTRKITLPYTGNYENLQTAAHKIRENIDLGLPALSSAIT
TLFYYNAQSAPSALLVLIQTTAEAARFKYIERHVAKYVATNFKPNLAIISLENQWSALSKQIFLAQNQG
GKFRNPVDLIKPTGERFQVTNVDSDVVKGNIKLLLNSRASTADENFITTMTLLGESVVEFPWVPGVGVP
GVGALWKTMLKELGTMALHAGKAALGAAADTISQGTQ* 33
MALWKTMLKELGTMALHAGKAALGAAADTISQGTQVPGVGVPGVGGSDVNFDLSTATAKTYTKFIEDFR
ATLPFSHKVYDIPLLYSTISDSRRFILLDLTSYAYETISVAIDVTNVYVVAYRTRDVSYFFKESPPEAY
NILFKGTRKITLPYTGNYENLQTAAHKIRENIDLGLPALSSAITTLFYYNAQSAPSALLVLIQTTAEAA
RFKYIERHVAKYVATNFKPNLAIISLENQWSALSKQIFLAQNQGGKFRNPVDLIKPTGERFQVTNVDSD
VVKGNIKLLLNSRASTADENFITTMTLLGESVVEFPW* 34
VPGVGVPGVGKWCFRVCYRGICYRRCRVPGVGVPGVGGATGSDVNFDLSTATAKTYTKFIEDFRATLPF
SHKVYDIPLLYSTISDSRRFILLNLTSYAYETISVAIDVTNVYVVAYRTRDVSYFFKESPPEAYNILFK
GTRKITLPYTGNYENLQTAAHKIRENIDLGLPALSSAITTLFYYNAQSAPSALLVLIQTTAEAARFKYI
ERHVAKYVATNFKPNLAIISLENQWSALSKQIFLAQNQGGKFRNPVDLIKPTGERFQVTNVDSDVVKGN
IKLLLNSRASTADENFITTMTLLGESVVNVPGVGVPGVGHGVSGHGQHGVHG 35
VPGVGVPGVGFLPLLAGLAANFLPTIICFISYKCVPGVGVPGVGGATGSDVNFDLSTATAKTYTKFIED
FRATLPFSHKVYDIPLLYSTISDSRRFILLNLTSYAYETISVAIDVTNVYVVAYRTRDVSYFFKESPPE
AYNILFKGTRKITLPYTGNYENLQTAAHKIRENIDLGLPALSSAITTLFYYNAQSAPSALLVLIQTTAE
AARFKYIERHVAKYVATNFKPNLAIISLENQWSALSKQIFLAQNQGGKFRNPVDLIKPTGERFQVTNVD
SDVVKGNIKLLLNSRASTADENFITTMTLLGESVVNVPGVGVPGVGKLAKLAK KLAKLAK
[0141] The G.I. tract of shrimps and prawns consists of the
proventriculus or gastric mill, digestive gland, midgut and its
diverticula, and the rectum. Two distinct cell types occur in the
digestive gland, a secretory type, and a
mucopolysaccharide-containing type, whose function is not clear.
The digestive gland has no intrinsic muscles, and depends on
extrinsic muscles, and possibly ingested water, for filling and
emptying. The midgut or hepatopancreas extends to the sixth
abdominal somite and faecal material is contained in a peritrophic
membrane. Defecation was at a peak 5-8 hr after food ingestion, but
continued up to 20 hr. The rectum appeared to have the additional
function of pumping water into the gut via the anus. As the pH of
seawater is around 8, it is natural for trypsin and chymotrypsin to
be the main physiological digestive enzymes whose pH optima is pH 8
unlike pepsin whose optima is at pH 2. Thus, pepsin was absent from
the digestive tract of shrimps. The ability of RetroMAD1 to survive
digestion by trypsin and chymotrypsin allowed it to be an effective
therapeutic protein that could be administered along with the feed
pellets itself thereby enabling a mode of delivery that is
practical for shrimp farmers to employ. The results are shown in
Table 3 above. Conjugating these peptides with MAP30, surprisingly
render the fusion protein stable for oral administration as shown
in its ability to survive protease digestion.
Example 6
Trial using Orally Delivered RetroMAD1 and an Immunostimulant
Beta-Defense on Asian Seabass
[0142] Between 27 Apr. 2012 to 23 May 2012, a 4-week trial was
carried out by Temanse Aquaculture S/B at its Sekayu Nursery centre
in Malaysia where survivor Asian Sea Bass Lates calcarifer
juveniles suffering from an unknown disease syndrome which was VNN
and Iridovirus PCR-'ve were treated with individual regimes of
RetroMAD1 and Beta-Defense (a commercial immune-stimulant) and a
combination regime, against an untreated control. From a shipment
of 30,000 fingerlings from Singapore, 20,000 quickly died within a
matter of days and 240 similar sized apparently healthy animals
were selected from the 10,000 survivors and were placed in 4
aquariums measuring 40.times.60.times.100cm each with 60
fingerlings for 3 days acclimation. Some minor mortalities occurred
during acclimation and the experiment began with 50 fishes per
batch. All water quality parameters such as Dissolved Oxygen, pH,
ammonia and nitrite are regularly measured to ensure these were
within normal acceptable ranges. The data is presented in Table 5
below.
TABLE-US-00009 TABLE 5 Results of experiment Lates calcarifer
survivor juveniles Weight gain and FCR Survival Rate % Initial
Final Treatment Day 1 Day 6 Day 14 Day 22 Day 28 Weight Weight FCR
Control - feed only 100 0 0 0 0 5.7 7.53 n.a. Treatment - feed + BD
100 0 0 0 0 5.71 7.22 n.a. Treatment - feed + RetroMAD1 100 100 100
48 48 5.65 15.8 1.8 Treatment - feed + BD + RetroMAD1 100 100 100
78 78 5.67 17.9 0.5 Carried out at Sekayu Nursery, Kuala Berang,
Malaysia belonging to Temanse Aquaculture S/B
[0143] All control and Beta-Defense fishes died on day 6. No more
mortalities occurred again until on day 22 and by day 28 at the end
of the experiment, the RetroMAD1 treatment had 48% survival and the
fingerlings had grown from 5.65 g-15.8 g mean weight with an FCR of
1.8. The combination treatment of RetroMAD1 gave a very convincing
78% survival with growth from 5.67-17.9 g mean weight with an FCR
of 0.5. Beta-Defense was given at 45 ml per 300 g of pellet feed
while RetroMAD1 was given at 0.1 ml diluted with 25 ml distilled
water and added to the 300 g of feed. In the combination treatment,
both were given together to 300 g of feed. Although the primary
pathogen has not yet been determined, there was evidence of some
secondary bacterial infection. We suspect however, that the primary
pathogen is viral in nature as RetroMAD1 is a broad-spectrum
antiviral oral-delivery protein drug. The efficacy shown with the
78% survival over 4 weeks also indicates that RetroMAD1 is
efficacious in fishes also. Thus, there is every potential to use
this method also in the production of SPF eggs and fingerlings in
fish breeding.
Example 7
The Stability of RetroMAD1, RetroGAD1, Amatilin and Tamapal1
[0144] The polypeptides, RetroMAD1, RetroGAD1, Amatilin and
Tamapal1 were capable of going through various thermocycler
protocols that mimic post-extrusion processing temperatures in
making extruded shrimp feed coated with RetroMAD1 and then coated
again with a marine edible oil.
[0145] The fusion peptide solutions to be tested were loaded using
a micropipette into 0.2 ml PCR tubes that were then placed into a
thermocycler (Labnet International, MultiGene Gradient) which was
then programmed to run at various temperature regimes as mentioned
in Table 6. Each regime was made up of a short high temperature
phase of 15 minutes followed by a longer medium temperature phase
of 45 minutes. These were to mimic the actual temperature
conditions when an extruded feed in the form of a wafer shaped
pellet left the extrusion barrel of a twin-screw extruder which in
this case is a Clextral BC45. The wafer was then sprayed with
sufficient squid oil post extrusion as to form an external lipid
barrier. In these thermocycler trials, the harshest condition was a
15 minute 70.degree. C. exposure followed by a 45 minute 55.degree.
C. exposure. Samples were then run on SDS-PAGE with the lanes as
follows:--Lanes: M, marker; 1, negative control treated with
2.times. .beta.-mercaptoethanol positive loading dye; 2, negative
control treated with 2.times. .beta.-mercaptoethanol negative
loading dye; 3, Sample subjected to the temperature regime and
treated with 2.times. .beta.-mercaptoethanol positive loading dye;
4, sample subjected to the temperature regime and treated with
2.times. .beta.-mercaptoethanol negative loading dye; 5, sample
subjected to the temperature regime and treated with 2.times.
.beta.-mercaptoethanol positive loading dye; 6, Sample subjected to
the temperature regime and treated with 2.times.
.beta.-mercaptoethanol negative loading dye. Comparison of the gel
bands against the control gave a physical evidence as to whether
the protein was damaged by the heat treatment or not.
TABLE-US-00010 TABLE 6 Parameters of temperature fluctuations.
Parameters Round 1/T1 Round 2/T2 Round 3/T3 Round 4/T4 Temperature
60 50 55 45 50 40 70 55 (.degree. C.) Time (mins) 15 45 15 45 15 45
15 45
[0146] As can be seen in FIG. 6, All four drugs, RetroMAD1 (A1 and
A2), RetroGAD1 (B1 and B2), Amatillin (C1 and C2) and Tamapal1 (D1
and D2) are intact under all treatments despite the presence of
BME. This indicates that all four drugs are stable and will not be
affected by the change in temperature.
Example 8
The Antiviral Activity of Peptides (Subjected to Various
Temperature Fluctuations using Thermocycler) against HSV-2
[0147] Amatilin, RetroGAD1 and Tamapal1 as described in Example 7,
were exposed to four sets of temperature fluctuations (T1, T2, T3
and T4) using thermocycler (Table 6 of Example 7). After exposure
to various temperature fluctuations, the peptides were subjected to
antiviral assay against HSV-2.
[0148] The cytotoxic activity of the peptides was quantified using
MTS-based cell titer 96 non-radioactive cell proliferation assay.
Briefly, monolayer cultures of Vero cells were exposed to
increasing concentrations of all the three peptides for 24, 48 and
72 h of incubation. After the incubation period, the maximal
concentration of the extract that did not exert toxic effect which
was regarded as the maximal non-toxic concentration (MNTD) was
determined using MTS assay.
[0149] After exposure to various temperature fluctuations using
thermocycler (Table 6), the antiviral activity of Amatilin,
RetroGAD1 and Tamapal1 was evaluated by simultaneous treatment. For
simultaneous treatment the mixture of the respective peptide and
virus inoculated onto Vero cells in 24-well culture plates and
incubated for 24, 48 and 72 h at 37.degree. C. under 5% CO.sub.2
atmosphere. At the end of the time period the samples were
harvested and viral DNA was extracted. The eluted DNA was then
subjected to RT-PCR.
[0150] The results obtained suggest that all the three peptides
were thermal stable. Amatilin and Tamapal1 showed the strongest
inhibitory activity against HSV-2 at all the four set of
temperature fluctuations (Table 7 and FIG. 7).
TABLE-US-00011 TABLE 7 Percentage of viral reduction caused by
Amatilin, RetroGAD1 and Tamapal1 exposed to various temperature
fluctuations in simultaneous treatment determined by PCR. Set of
temperature Peptides fluctuations Amatilin RetroGAD1 Tamapal1 T1
97.28 94.88 86.21 T2 94.05 96.95 90.36 T3 97.85 63.04 97.64 T4
86.00 75.91 93.65
Example 9
Leaching Rate of RetroMAD1, RetroGAD1, Amatilin and Tamapal1 from
the Wafer Pellets Produced in a Pilot-Scale Manufacture
[0151] The leaching rate study for as the various fusion protein
drugs as described in Example 7, was to study the time points when
RetroMAD1, RetroGAD1, Amatilin and Tamapal1 were leached out from
the wafers. Wafers containing the drugs were placed within in 30
ppt sea salt water in 1:100 weight to volume ratio. Shrimp wafer
pellets were formed by extrusion using a Clextral BC45 twin-screw
extruder that was sprayed post extrusion with the fusion protein
drugs to be tested followed by a spray coating in a vacuum chamber
with squid oil to serve as an outer hydrophobic layer to `lock-in`
the test drug as well as to serve an a feeding attractant for the
shrimp. Addition of RetroMAD1 was added at the rate of 300 mg/kg of
wafer pellets. At 0, 30, 60, 120 and 240 minutes, sea salt water
was collected to determined the concentration of the fusion protein
drugs that was leached out of the wafers into the sea salt water.
Capture ELISA (Promega, Glomax Multidetection System) was used to
determine the concentration of RetroMAD1, while Direct ELISA was
used for RetroGAD1, Amatilin and Tamapal1. In Capture ELISA, a 96
U-bottom well plated was coated with 1:1000 of rabbit
anti-RetroMAD1 antibody and was incubated at 4.degree. C.
overnight. The plate was then washed with PBS-Tween20 six times
before adding the samples collected at time point 0, 30, 60, 120
and 240 minutes and incubated at 37.degree. C. for an hour.
Subsequently, 1:2500 human anti-RetroMAD1 antibodies were added to
capture RetroMAD1 from the samples bound on the rabbit
anti-RetroMAD1 antibody. While in direct ELISA, a 96 well
U-bottomed plate was coated with the samples collected at time
point 0, 30, 60, 120 and 240 minutes and incubated overnight at
4.degree. C. The plate was then washed with PBS-Tween20 and added
with 1:500 rabbit antibodies against RetroGAD1, Amatilin and
Tamapal1 to capture the protein drug bound on the plate.
Subsequently, 1:10000 anti-rabbit IgG were added to detect rabbit
antibodies bind against the protein drugs. Absorbance was read at
490 nm and 600 nm. A standard curve of drug concentration against
absorbance was plotted to determine the concentration of the drug
in each sample.
[0152] Both RetroMAD1 and Tamapal1 began leaching out only after
120 minutes. Both Amatilin and RetroGAD1 did not show any signs of
leaching even 240 minutes. This shows that since shrimp usually
consume all their feed within 30-60 minutes, this method of oral
administration of these fusion protein drugs is viable for the
treatment of shrimp viruses. Furthermore, as shrimp digestion is
trypsin rather than chymotrypsin dependent, it does not matter that
the drug is presented along with the feed.
[0153] Antibodies toward RetroMAD1, RetroGAD1, Amatilin and
Tamapal1 were raised in 4 rabbits respectively. In each
immunization, rabbits were immunized with RetroMAD1, RetroGAD1,
Amatilin and Tamapal1 in single dose of 0.6 ml per rabbit which is
a dose of 0.2 mg/kg body weight for RetroMAD1, 0.9 ml per rabbit
which is a dose of 0.25 mg/kg body weight for RetroGAD1, 0.8 ml per
rabbit which is a dose of 0.25 mg/kg body weight for Amatilin and 1
ml per rabbit which is a dose of 0.25 mg/kg body weight for
Tamapal1.
[0154] Prior to immunization, on Day 0, blood was drawn from the
rabbits. Pre-bleed blood collected on Day 0 was used as the base
line in determining the antibody titer in rabbit. After
pre-bleeding the rabbit, first immunization was given according to
the dosage per body weight as mentioned above. Rabbits were bled
before giving another immunization on Day7, Day 14, Day 28 and Day
35. Blood serum of rabbits collected on Day7, Day 14, Day 28 and
Day 35 was used to determine antibody titer against RetroMAD1,
RetroGAD1, Amatilin and Tamapall. On Day 38, antibody towards
RetroMAD1, RetroGAD1, Amatilin and Tamapal1 raised in rabbits were
harvested. In harvesting the rabbit antibody, before bleeding, each
rabbit was given anesthesia (Ketamine and Xylazine) intravenously;
the sedative dose was calculated using the following formula
Ketamine=(30.times.body weight of the rabbit)/(Concentration of
Ketamine, 100 mg/ml)
Xylazine=(3.times.body weight of the rabbit)/(Concentration of
Xylazine, 20 mg/ml)
[0155] 50 ml of blood was collected from each rabbit. Blood was
centrifuged at 4000 rpm for 15 minutes; blood serum containing
antibody towards RetroMAD1, RetroGAD1, Amatilin and Tamapal1 was
collected and kept in -20.degree. C. for further use.
[0156] A direct ELISA was used to determine antibody titer in
rabbit serum. A 96-well U-bottomed plate was coated with 1 .mu.g/ml
of RetroMAD1, RetroGAD1, Amatilin and Tamapal1 in coating buffer
(0.2 M sodium carbonate-bicarbonate, ph 9.6). The sample coated
plate was incubated at 4.degree. C. overnight. Plates were washed
six times with 0.05% Tween-20 in PBS 1.times.. 100 .parallel.l of
1/10 rabbit serum was added to the well, a 1/2 serial dilution of
the rabbit serum was made. Rabbit serum was diluted in 1/10, 1/20,
1/40, 1/80, 1/160, 1/320, 1/640, 1/1280, 1/2560, 1/5120 and 1/10240
to determine the antibody titer. After incubation at 37.degree. C.
for 1 hour, plates were washed similarly and 100 .mu.l/well of
anti-rabbit IgG diluted 1:10000 in 5% BSA in PBS was added. After
incubation at 37.degree. C. for 1 hour, plates were washed and 100
.mu.l/well streptavidin-HRP diluted 1:20000 in 5% BSA in PBS was
added. After incubation at 37.degree. C. for 1 hour in the dark,
plates were washed and 100 .mu.l/well of OPD added to each well.
Plates were incubated in the dark for 30 min at room temperature
and reaction stopped with 50 .mu.l/well of 4N H.sub.2SO.sub.4.
Optical densities (OD) were measured at 490 nm and 600 nm as
background. The results are shown in FIG. 8A-D and tables 8-11.
TABLE-US-00012 TABLE 8 Concentration of RetroMAD1 leached out
against time Time (minutes) Concentration of RetroMAD1 (.mu.g/ml)
30 0 60 0 120 0 240 1
TABLE-US-00013 TABLE 9 Concentration of RetroGAD1 leached out at 0,
30, 60, 120 and 240 minutes Concentration of RetroGAD1 Time
(Minutes) (.mu.g/ml) 0 0.00000 30 0.00000 60 0.00000 120 0.00000
240 0.00000 Control 0.00000
TABLE-US-00014 TABLE 10 Concentration of Amatilin leached out at 0,
30, 60, 120 and 240 minutes Time (Minutes) Concentration of
Amatilin (.mu.g/ml) 0 0.00000 30 0.00000 60 0.00000 120 0.00000 240
0.00000 Control 0.00000
TABLE-US-00015 TABLE 11 Concentration of Tamapal1 leached out at 0,
30, 60, 120 and 240 minutes Time (Minutes) Concentration of
Tamapal1 (.mu./ml) 0 0.000000 30 0.000000 60 0.000000 120 0.000000
240 0.040167 Control 0.000000
Example 10
Short-Term Pharmacokinetics of RetroMAD1 in Shrimp using Capture
ELISA
[0157] In the short-term feeding study, shrimps were fed with 0.06
g of shrimp wafer pellets containing RetroMAD1 at an inclusion of
300 mg/kg. This study is to determine the short term kinetics of
RetroMAD1 in terms of absorption, retention and excretion. Shrimp
wafer pellets were formed by extrusion using a Clextral BC45
twin-screw extruder that was sprayed post extrusion with the fusion
protein drugs to be tested followed by a spray coating in a vacuum
chamber with squid oil to serve as an outer hydrophobic layer to
`lock-in` the test drug as well as to serve an a feeding attractant
for the shrimp. Addition of RetroMAD1 was added at the rate of 300
mg/kg of wafer pellets.
[0158] Healthy specimens of the commonly cultured Pacific white
shrimp Penaeus vannamei were selected from a shrimp farm in Tawau,
Sabah, Malaysia and a single specimen ranging from 2.4-5.8 g was
placed in each transparent plastic aquarium tank of 10 litres total
capacity containing 5 litres of seawater at 32 parts per thousand
salinity. Specimens were acclimated for a week prior to the
experiment and 50% water was changed daily by siphoning. A single
airstone was provided such that aeration was sufficiently provided
such that the animal did not display any signs of being stressed. A
plastic netting was provided on top to prevent the specimens from
jumping out. For each sampling time point, tanks were present in
triplicate as in Group 1, 2 and 3. As there were 8 sampling time
points, 24 tanks were prepared as shown in the Table 12.
TABLE-US-00016 TABLE 12 Experiment design for measuring short-term
pharmacokinetics of RetroMAD1 in shrimp Sampling Points Number of
Shrimp per tank Time (Hours) Group 1 Group 2 Group 3 Control 1 1 1
0.5 1 1 1 1 1 1 1 1.5 1 1 1 2 1 1 1 3 1 1 1 5 1 1 1 8 1 1 1
[0159] At each sampling time point, the feces were collected by
siphoning, the shrimp dissected removing the hepatopancreas well as
the muscle of the last abdominal segment of the tail which was
stored in PBS buffer and stored at -40.degree. C. Note that the
Control were fed normal shrimp pellets without RetroMAD1. The
shrimp were unfed for the duration of the experiment after
completely ingesting the test and control feeds. The weights of the
feces, hepatopancreas and tail muscle (only the last abdominal
segment) collected are presented in the table 13 below.
TABLE-US-00017 TABLE 13 Weight of each shrimp, hepatopancreas, tail
muscle (last segment only) and feces Captured ELISA (Promega,
Glomax Multidetection System) was used to determine concentration
of RetroMAD1 in the samples. The tail muscle sampled was in the
last abdominal segment after the anus to ensure any result did not
come from the GI tract. In captured ELISA, a 96 U-bottom well
plated was coated with 1:1000 of rabbit anti- RetroMAD1 antibody
(as mentioned in Example 9) and was incubated at 4.degree. C.
overnight. Plate was then washed with PBS-Tween20 six times before
adding the samples of hepatopancreas, tail muscle and feces and
incubated at 37.degree. C. for an hour. Subsequently, 1:2500 human
anti-RetroMAD1 antibody was added to capture RetroMAD1 from the
samples bound on the rabbit anti-RetroMAD1 antibody. Absorbance was
read at 490 nm and 600 nm. A standard curve of concentration of
RetroMAD1 (.mu.g/ml) against absorbance as shown in Table 13 was
plotted to determine the concentration of RetroMAD1 in each sample.
Weight (grams) Time Tail (Hour) Tank Whole Shrimp Hepatopancreas
Muscle Feces 0 (Control) 1 3.238 0.120 0.108 0.000 2 3.227 0.160
0.127 0.038 3 3.554 0.190 0.231 0.043 0.5 1 3.600 0.250 1.960 0.045
2 2.440 0.170 0.142 0.040 3 3.720 0.187 0.136 0.020 1 1 2.785 0.152
0.100 0.065 2 3.213 0.141 0.133 0.030 3 4.236 0.211 0.208 0.015 1.5
1 2.130 0.126 0.095 0.070 2 4.117 0.175 0.206 0.053 3 1.612 0.100
0.083 0.086 2 1 5.500 0.236 0.222 0.041 2 2.784 0.155 0.116 0.052 3
2.993 0.182 0.160 0.056 3 1 3.538 0.190 0.142 0.024 2 3.719 0.175
0.154 0.083 3 3.995 0.199 0.147 0.054 5 1 3.508 0.154 0.188 0.060 2
3.962 0.194 0.200 0.016 3 2.443 0.155 0.104 0.050 8 1 4.995 0.245
0.245 0.035 2 5.840 0.182 0.292 0.034 3 3.460 0.148 0.146 0.038
[0160] Table 14 and FIG. 9 show that hepatopancreal absorption of
RetroMAD1 was detectable at 1.5 hours post-feeding and peaked at 5
hours post-feeding while RetroMAD1 was detectable in the tail
muscle as early as 3 hours post-feeding.
TABLE-US-00018 TABLE 14 Concentration of RetroMAD1 against time
Treated with RetroMAD1 Time (hours) Hepatopancreas Tail muscle
Feces Control 0.5 0 0 0 0 1 0 0 0 0 1.5 0 0 0 0 2 1.133 0 0 0 3
1.983 0 0 0 5 3.867 0.9333 0 0 8 2.717 1.783 0 0
Example 11
Long-Term Pharmacokinetics of RetroMAD1 in Shrimp using Capture
ELISA
[0161] In the long-term feeding study, shrimps were fed with 0.2 g
of shrimp pellets containing RetroMAD1 at 300 mg/kg inclusion rate.
This study is to further determine the pharmacokinetics of
RetroMAD1 in terms of absorption, retention and excretion over 7
days.
[0162] In this study, Healthy specimens of the commonly cultured
Pacific white shrimp Penaeus vannamei were selected from a shrimp
farm in Tawau, Sabah, Malaysia and 8 pcs of 10.0 g +/-0.5 g
specimen were placed in a single transparent plastic aquarium tank
of 50 litres total capacity containing 40 litres of seawater at 32
parts per thousand salinity. Specimens were acclimated for a week
prior to the experiment and 50% water was changed every alternate
day by siphoning. A single airstone was provided such that aeration
was sufficiently provided such that the animal did not display any
signs of being stressed. A plastic netting was provided on top to
prevent the specimens from jumping out. Feces were collected daily
by siphoning and placed into a 1.5 ml plastic tube that was capped
and stored at -40.degree. C. until required. For each sampling time
point, tanks were present in triplicate as in Group 1,2 and 3. As
there were 7 sampling time points as well as one control, 24 tanks
were prepared each with 8 specimens as shown in Table 15:
TABLE-US-00019 TABLE 15 Experiment design for measuring long-term
pharmacokinetics of RetroMAD1 in shrimp Sampling Points Number of
Shrimps per tank Time (Days) Group 1 Group 2 Group 3 Control
Control 1 Control 2 Control 3 1 8 8 8 2 8 8 8 3 8 8 8 4 8 8 8 5 8 8
8 6 8 8 8 7 8 8 8
[0163] Within the first day, there were 6 sampling points at 2, 4,
6, 8, 12 and 16 hours. As there were 8 animals, one was removed at
each sampling point and the feces and dissected hepatopancreas as
well as the tail muscle from the last abdominal segment pooled and
kept in PBS at -40.degree. C. till required. The sampling point at
24 hours was taken at the start of Day 2 while the 36 hours
sampling point taken mid-way through Day 2. Thereafter, there were
sampling points daily. Captured ELISA (Promega, Glomex
Multidetection System) was used to determine concentration of
RetroMAD1 in hepatopancreas, tail muscle and faeces, in the method
previously described earlier.
[0164] Table 16 and FIG. 10 showed that hepatopancreal RetroMAD1
peaked after 5 hours in agreement with the previous experiment
(Example 10). Residual RetroMAD1 could be detected up to 144 hours
(day 6) even after faecal RetroMAD1 was no long detectable after 72
hours (day 3). Detectable RetroMAD1 in the tail muscle remained
only for 8 hours showing that the drug residue is quickly broken
down by active swimming shrimp.
[0165] This indicated a safe `withdrawal period` of 14 days for
head-on shrimp product (sold with the head containing the
hepatopancreas) and 7 days for a headless shrimp product (where the
head containing the hepatopancreas is removed at the processing
factory prior to freezing.
TABLE-US-00020 TABLE 16 Concentration of RetroMAD1 in
hepatopancreas, tail muscle and feces at 2, 4, 6, 8, 10, 12, 18,
24, 36, 48, 72, 96, 120, 144 and 168 hours. Concentration of
RetroMAD1 (.mu.g/ml) Time (Hours) Hepatopancreas Tail Muscle Feces
Control 2 189.62 0 0 0 4 335.085 5.272 115.751 0 6 304.758 16.015
151.794 0 8 209.824 0 238.178 0 10 174.754 0 234.733 0 12 168.082 0
224.924 0 18 165.378 0 256.926 0 24 160.538 0 194.13 0 36 147.025 0
195.96 0 48 136.772 0 201.383 0 72 75.827 0 0 0 96 49.567 0 0 0 120
40.343 0 0 0 144 3.275 0 0 0 168 3.426 0 0 0
Example 12
An Orally Administered WSSV Challenge to Post-Larvae Treated with
Various Fusion Proteins
[0166] Protein concentrations were determined by Bradford Analysis
(Quick Start Bradford Protein Assay,
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110065A.pdf).
The post-larvae were acclimatized for 1 week, continued with
pre-infection period of 3 weeks and post-infection period of 1
week. The post-larvae were fed throughout the duration. Feeding was
done 3 times a day mixed with the drugs at a ratio of 150 ml/kg
feed. RetroMAD1 and Tamapal1 were effective against the WSSV oral
infection as shown in Tables 16-19. No mortality was observed until
Day 30.
[0167] Feed was observed to be completely consumed justifying the
increase in feeding rate over the period of the experiment.
Tamapal1 differs from Tamapal1 (A) by use of a different refolding
buffer.
[0168] Tail muscle tissue (20 mg) was used to determine the viral
number retained in the shrimp. DNA extraction was carried out using
a `salting-out` method (Miller, S. A.; Dykes, D. D.; Polesky, H. F.
(1988). "A simple salting out procedure for extracting DNA from
human nucleated cells". Nucleic acids research 16 (3): 1215).
Tissues were lysed in 600 .mu.l of lysis buffer (25 mM EDTA, 2%
SDS) containing 1.5 .mu.l of 20 mg/ml proteinase K. The lysate was
incubated at 65.degree. C. for 45 minutes or until the solid tissue
have been completely dissolved. The lysate was treated with 1.5
.mu.l of 4 mg/ml RNase A for another 15 minutes followed by cooling
to room temperature for 5 minutes. Protein precipitation solution
(200 .mu.l) was added and the lysate was vortexed for 25 seconds
followed by incubation on ice for 5 minutes. The homogenate was
centrifuged at 13,000.times.g got 10 minutes and 600 .mu.l of
supernatant was transferred to a 1.5 ml centrifuge containing 600
.mu.l of isopropanol. The tube was inverted gently 40 times. DNA
was precipitated at 13,000.times.g for 5 minutes, the supernatant
was discarded. The DNA was further washed by adding 600 .mu.l of
70% ethanol and centrifuged for another 3 minutes. Finally the
supernatant was discarded and the tube was left to air-dry for 15
minutes. TE buffer was added at 100 .mu.l and used for PCR.
[0169] In this study, three sense primers (F1 [SEQ ID NO:40]: 5'
ATGGATTTGGCAACCTAACA 3', F2 [SEQ ID NO:41]: 5' AATTCGTGGAGAGAGGTCC
3' and F3 [SEQ ID NO:42]: 5' ATCTCTACCGTCACACAGCC 3') and 1
antisense primer (R1 [SEQ ID NO:43]: 5' GAAGATTTTAATGTCCTTGCTCG 3')
were designed from the nucleotide sequence of a 626 by WSSV viral
product (van Hulten M. C. W., Witteveldt J., Peters S.,
Kloosterboer N., Tarchini R., Fiers M., Sand brink H., Klein
Lankhorst R. and Vlak J. M. 2001. The white spot syndrome virus DNA
genome sequence. Virol 67: 233-241).
[0170] The F1 and R1 primers were used as external primers to
generate a primary PCR product of 500 base pairs (bp) while F2 and
F3 were used internal primers along with R1 to generate nested PCR
product of 300 and 200 bp, respectively. The 30 .mu.l PCR reaction
contained 50 mM KCI, 10 mM Tris-HCl, pH 9.0, 1.5 mM MgCl, 0.2 mM
each of deoxy (d) ATP, dCTP, dGTP and dTTP, 1 .mu.M of RI, 0.3
.mu.M of F1, 0.3 .mu.M of F2 and 0.4 .mu.M of F3, 1.25 U Taq
polymerase. The PCR reaction initiated by heating the mixture
95.degree. C. for 5 min followed by 30 cycles of 30 s at 95.degree.
C., 30 s at 55.degree. C. and 30 s at 72.degree. C. with a final
extension of 10 min at 72.degree. C. Following PCR, the amplified
products were analyzed by electrophoresis in 2% agarose gel stained
with ethidium bromide and visualized by ultraviolet
transillumination.
TABLE-US-00021 TABLE 16 Post larvae mortality rate. Mortality
(times) 24 48 72 96 120 144 168 Tanks hours hours hours hours hours
hours hours Control (+) 1 0 6 9 10 10 10 10 Control (+) 2 0 2 10 10
10 10 10 Tamapal1 1 0 2 8 9 9 9 9 Tamapal1 2 0 0 0 0 0 0 0 Tamapal1
(A) 1 0 3 9 9 9 9 9 Tamapal1 (A) 2 0 5 8 10 10 10 10 RetroGAD1- 1 0
2 10 10 10 10 10 RetroGAD1- 2 0 1 8 10 10 10 10 Amatilin 1 0 4 10
10 10 10 10 Amatilin 2 0 4 10 10 10 10 10 RetroMAD1-1 0 0 0 0 0 0 0
RetroMAD1-2 0 0 0 0 0 0 0 Control (-) 1 0 0 0 0 0 0 0 Control (-) 2
0 0 0 0 0 0 0
TABLE-US-00022 TABLE 17 PCR result at the end of trial period.
(Each set of drugs were tested in duplicates.) Pcs Tanks 1 2 3 4 5
6 7 8 9 10 Control (+) 1 H H H H M H H H H H Control (+) 2 H H H H
H H H H H H Tamapal1 1 H H H H H H H H H Tamapal1 2 -- -- -- -- --
-- -- -- -- -- Tamapal1 (A) 1 H H M H H M H H H H Tamapal1 (A) 2 H
H H H H M L L H H RetroGAD1-1 H H M H H H H H H H RetroGAD1-2 H H H
H H H H H H H Amatilin 1 H H H M H H H H H H Amatilin 2 H H H M H H
M H M H RetroMAD1-1 -- -- -- -- -- -- -- -- -- -- RetroMAD1-2 -- --
-- -- -- -- -- -- -- -- Control (-) 1 -- -- -- -- -- -- -- -- -- --
Control (-) 2 -- -- -- -- -- -- -- -- -- -- H: High; M: Medium; L:
Low; and --: no band observed.
TABLE-US-00023 TABLE 18 Concentration of drugs used. Concentration
(mg/ml) Tamapal1 0.468 Tamapal1 (A) 1.43 RetroGAD1 0.58 Amatilin
0.615 RetroMAD1 1.14
TABLE-US-00024 TABLE 19 Feeding rate from Day 1-30. Amount of drug
in .mu.g ingested by a test animal Pre & post Feed/day/
assuming even feeding ** infection aquarium* Tamapal1 Tamapal1 (A)
RetroGAD1 Amatilin RetroMAD1 Day 1-4 0.09 g 0.63 1.9305 0.783
0.83025 1.539 Day 5-9 0.12 g 0.8424 2.574 1.044 1.107 2.052 Day
10-16 0.18 g 1.2636 3.861 1.566 1.6605 3.078 Day 17-30 0.27 g
1.8954 5.7915 2.349 2.4907 4.617 *10 pieces of post larvae per
aquarium ** Even feeding assumes that all test animals ingested at
the same rate.
Example 13
Amatilin having Antibacterial Activity on Vibrio cholera and Vibrio
parahaemolyticus from In Vitro Assay
[0171] The minimum inhibitory concentration (MIC) of Amatilin
against Vibrio cholera and Vibrio parahemolyticus was performed
according to the Clinical and Laboratory Standard Institute
guidelines using the broth micro dilution method. Stock Amatilin at
concentration of 1290 .mu.g/ml was two-fold serially diluted in
cationically adjusted Mueller Hinton broth (CAMHB) in 96 well round
bottom plate to 50 .mu.l. Bacterial cultures from -80.degree. C.
glycerol stock were passaged twice on nutrient agar and resuspended
in phosphate buffered saline (PBS) to OD.sub.625 0.08-0.1 which was
equivalent to 1-2.times.10.sup.8 cfu/ml. The suspension was
adjusted to 1.times.10.sup.6 cfu/ml and added in equal volume (50
.mu.l) to the 96 well plate prepared with the serial dilutions of
Amatilin. Final testing concentration of Amatilin ranged from 2.52
.mu.g/ml to 322.5 .mu.g/ml. Plates were incubated for 18-24 hours
under 37.degree. C. MIC was read as the lowest concentration of
drug that completely inhibits the visible growth of the bacteria.
Following that, aliquot of 10 .mu.l from each well was serially
ten-fold diluted in PBS and plated on Mueller Hinton agar for
viable colony count. Wells with bacterial density of more than
1.times.10.sup.10 cfu/ml was noted as ">1.times.10.sup.10
cfu/ml", implying no
TABLE-US-00025 Cell count 24 hr Concentration Actual cell Bacteria
(microgram/ml) Time (hr) Cell counted.sup.a Dilution factor no.
(cfu/ml).sup.b Vibrio cholerae Untreated 0 188 10.sup.3 1.88
.times. 10.sup.5 Untreated 24 Overcrowded 10.sup.10 >1 .times.
10.sup.10 322.5 24 0 10.sup.2 0 161.25 24 51 10.sup.2 5.1 .times.
10.sup.3 80.63 24 124 10.sup.9 >1 .times. 10.sup.10 40.31 24
Overcrowded 20.16 24 Overcrowded 10.08 24 Overcrowded 5.04 24
Overcrowded 2.52 24 Overcrowded Vibrio Untreated 0 26 10.sup.4 2.6
.times. 10.sup.5 parahaemolyticus Untreated 24 Overcrowded
10.sup.10 >1 .times. 10.sup.10 322.5 0 1.sup. 0 161.25 0 1.sup.
0 80.63 27 10.sup.8 2.7 .times. 10.sup.9 40.31 Overcrowded
10.sup.10 >1 .times. 10.sup.10 20.16 Overcrowded 10.sup.10 >1
.times. 10.sup.10 10.08 Overcrowded 10.sup.10 >1 .times.
10.sup.10 5.04 Overcrowded 10.sup.10 >1 .times. 10.sup.10 2.52
Overcrowded 10.sup.10 >1 .times. 10.sup.10 .sup.aPlates with
more than 200 colonies were denoted as "overcrowded". .sup.bActual
cell no. (cfu/ml) more than 1 .times. 10.sup.10 were denoted as
">1 .times. 10.sup.10".
inhibitory activity. Results as shown in FIGS. 11A and B and tables
20 and 21 were pooled from duplicate tests in three independent
trials.
TABLE-US-00026 TABLE 20 MIC of Amatilin Bacteria MIC (.mu.g/ml)
Vibrio cholerae 161.25 (10.08) Vibrio parahaemolyticus 161.25
(161.25)
Table 21: Antibacterial Effect of Amatilin on V. cholerae and V.
parahemolyticus.
Example 14
Antiviral Activity of Amatilin, RetroGAD1, RetroMAD1 and Tamapal1
using HSV2
Cytotoxicity of Tested Peptides on Vero Cells
[0172] The effect of Amatilin, RetroGAD1, RetroMAD1 and Tamapal1 on
the growth of Vero cells was examined to rule out any direct
cytotoxicity. Monolayer cultures of Vero cells were exposed to
increasing concentrations of Amatilin, RetroGAD1, RetroMAD1 and
Tamapall. After 24, 48 and 72 h of incubation, cell viability was
determined using MTS assay. Results shown indicate the accepted
maximal nontoxic concentrations of the four peptides on Vero cells.
At the chosen
[0173] Maximal non-toxic dose (MNTD) as shown in Table 22, the
peptides did not impair the cell viability with respect to the
untreated control group.
TABLE-US-00027 TABLE 22 Maximal non-toxic dose of the peptides on
Vero cells MNTD (.mu.g/ml) Peptide 24 h 48 h 72 h Amatilin 15 15 15
RetroGAD1 10 10 10 RetroMAD1 50 50 50 Tamapal1 15 15 15
The Antiviral Activity of Peptides against HSV-2
[0174] The antiviral activity of Amatilin, RetroGAD1, RetroMAD1 and
Tamapal1 was evaluated by simultaneous treatment. For simultaneous
treatment the mixture of the respective peptide and virus was
inoculated onto Vero cells and incubated for 24, 48 and 72 h at
37.degree. C. under 5% CO.sub.2 atmosphere. At the end of the time
period the samples were harvested and viral DNA was extracted. The
eluted DNA was then subjected to RT-PCR.
[0175] The results obtained suggested that all the four peptides
have strong inhibitory activity against HSV-2 via simultaneous
treatment at the maximal non-toxic dose (MNTD) (Table 22). Amatilin
showed 99.88, 99.99 and 99.98% of inhibition, respectively, after
24, 48 and 72 h. RetroGAD1 exhibited 95.45, 91.71 and 89.95%
inhibitory activity, respectively, at 24, 48 and 72 h.
[0176] RetroMAD1 showed 99.67, 99.96 and 99.87% of viral reduction,
respectively, at 24, 48 and 72 h. Tamapal1 showed 98.75, 98.00 and
98.98% inhibition, respectively, at 24, 48 and 72 h (Table 23 and
FIG. 12).
TABLE-US-00028 TABLE 23 Percentage of viral reduction caused by
Amatilin, RetroGAD1, RetroMAD1 and Tamapal1 in simultaneous
treatment at 72 h determined by PCR. Time Peptides 24 h 48 h 72 h
Amatilin 99.88 99.99 99.98 RetroGAD1 95.45 91.71 89.95 RetroMAD1
99.67 99.96 99.87 Tamapal1 98.75 98.00 98.98
Example 15
Effects of Temperature on the Stability of Amatilin, RetroGAD1 and
Tamapal1 Evaluated Via Antiviral Activity
[0177] Amatilin, RetroGAD1 and Tamapal1 were placed in -20, 4, 26
and 37.degree. C. for up to 30 days. The peptides were also placed
in 50.degree. C. for up to 7 days. To further investigate the
thermostability of the peptides, they were also exposed to four set
of various temperature fluctuations. Subsequently, the peptides
were analyzed for their antiviral activity against HSV-2 via
simultaneous treatment.
[0178] The antiviral activity of peptides (subjected to various
temperatures) against HSV-2 The antiviral activity of Amatilin,
RetroGAD1 and Tamapal1 after incubation at different temperatures
(-20, 4, 26, 37 and 50.degree. C.) for 1, 7 and 30 days was
evaluated by simultaneous treatment. For simultaneous treatment the
mixture of the respective peptide and virus inoculated onto Vero
cells and incubated for 24, 48 and 72h at 37.degree. C. under 5%
CO.sub.2 atmosphere. At the end of the time period the samples were
harvested and viral DNA was extracted. The eluted DNA was then
subjected to RT-PCR.
[0179] The results obtained suggest that all the three peptides
were thermal stable. The peptides exposed to various temperatures
for 1, 7 and 30 days showed strong inhibitory activity against
HSV-2 via simultaneous treatment at the maximal non-toxic dose
(MNTD) (Table 20 in Example 14). Amatilin was stable at high
temperatures, 26 and 37.degree. C. for up to 30 days giving 99.95
and 91.78% of inhibition, respectively. Amatilin was also stable at
50.degree. C. for up to 7 days with 94.75% inhibitory activity
(Table 24 and FIG. 13A). RetroGAD1 exhibited 99.01 and 78.52%
inhibitory activity, respectively, after incubation at 26 and
37.degree. C. for up to 30 days. The peptide showed 95.03% of viral
reduction after incubation at 50.degree. C. for up to 7 days (Table
24 and FIG. 13B). Tamapal1 was stable for up to 30 days at 26 and
37.degree. C. giving 88.12 and 91.78% inhibitory activity,
respectively. The peptide remained stable for 7 days at 50.degree.
C. with 99.42% of viral reduction (Table 24 and FIG. 13C).
TABLE-US-00029 TABLE 24 Percentage of viral reduction caused by
Amatilin, RetroGAD1 and Tamapal1 incubated at different
temperatures for 1, 7 and 30 days in simultaneous treatment
determined by PCR. Peptides Temperature Amatilin RetroGAD1 Tamapal1
(.degree. C.) Day 1 Day 7 Day 30 Day 1 Day 7 Day 30 Day 1 Day 7 Day
30 -20 99.84 98.00 99.98 95.93 98.94 98.96 98.73 94.77 91.01 4
89.35 99.98 99.92 99.66 98.92 98.30 95.84 92.92 90.87 26 94.53
99.75 99.95 99.77 96.24 99.01 98.49 92.92 88.12 37 98.55 91.45
91.78 95.54 95.61 78.52 99.45 99.16 91.78 50 94.31 94.95 -- 97.12
95.03 -- 91.36 99.42 --
Example 16
Method of Micronizing RetroMAD1 into a Free-Flowing Powder by
Supercritical Fluid Drying (SCFD)
[0180] A 10 L high pressure vessel of the configuration
conventionally used for Supercritical Fluid Drying (SCFD) was used
to dry and produce a micronized form of powdered free-flowing
RetroMAD1 under the conditions of 120 bar; 37 C; 300 kg/hr CO2 flow
that gave 88-89% yield in 2 cases and a lower 58% yield in one case
due to operator error. The resulting powder was observed to be
slightly cubic when viewed under Scanning Electron Microscope (SEM)
and about 1 micron in size on the average. This confirms that
RetroMAD1 may be efficiently manufactured as a powder for
incorporation into tablets, capsules and animal feed pellets
whether for terrestrial or aquatic application. The schematics of
the process is shown in FIG. 14 and an SEM picture showing the
morphology of the RetroMAD1 crystals is shown in FIG. 15.
Bioactivity of RetroMAD1 SCFD Micronized Powder Evaluated using
HSV-2
[0181] The bioactivity of the micronized form of powdered
free-flowing RetroMAD1 produced using
[0182] SCFD was tested via antiviral assay against HSV-2. For the
antiviral test, RetroMAD1 micronized powder was dissolved in two
different solvents: (i) ultra pure water with 5.5 mM NaOH; and (ii)
ultra pure water. Ultra pure water was produced using a Sastec
ST-WP-UVF machine.
Cytotoxicity of RetroMAD1 Micronized Powder
[0183] Prior to screening RetroMAD1 micronized powder for its
antiviral properties, it was subjected to cytotoxicity assay in
order to identify the maximal concentration, which could be
non-toxic to Vero cells. The cytotoxic activity of the peptides was
quantified using MTS-based cell titer 96 non-radioactive cell
proliferation assay. Briefly, monolayer cultures of Vero cells were
exposed to increasing concentrations of the dissolved RetroMAD1
powder for 24, 48 and 72h of incubation. After the incubation
period, the maximal concentration of the protein that did not exert
toxic effect is regarded as the maximal non toxic concentration
(MNTD) was determined using MTS assay.
[0184] Results as shown in Table 25 indicate that the accepted
maximal nontoxic concentrations of RetroMAD1 micronized powder on
Vero cells were less than 20 .mu.g/ml. At the chosen MNTD, the
peptides did not impair the cell viability with respect to the
untreated control group.
TABLE-US-00030 TABLE 25 Maximal non-toxic dose of RetroMAD1
micronized powder on Vero cells MNTD, ug/ml Peptide 24 h 48 h 72 h
RetroMAD1 powder (in water + NaOH) 15 15 15 RetroMAD1 powder (in
water) 5 5 5
The Antiviral Activity of RetroMAD1 Micronized Powder against
HSV-2
[0185] The antiviral activity of RetroMAD1 micronized powder was
evaluated by simultaneous treatment. For simultaneous treatment,
the mixture of RetroMAD1 and virus were inoculated onto Vero cells
in 24-well culture plates and incubated for 24 and 48h at
37.degree. C. under 5% CO.sub.2 atmosphere. At the end of the time
period the samples were harvested and viral DNA was extracted. The
eluted DNA was then subjected to RT-PCR.
[0186] The results obtained suggested that RetroMAD1 in powder form
exhibited strong inhibitory activity against HSV-2 via simultaneous
treatment giving between 85% -100% of inhibition. RetroMAD1 powder
was dissolved in ultrapure water with NaOH showed higher percentage
of viral reduction compared to the powder dissolved in ultroapure
water alone at the MNTD (Table 26 and FIG. 16). SCFD was therefore
a viable method of producing RetroMAD1 in a solid dose format good
for incorporation into tablets, capsules, medicated chewing gum and
aquatic feed pellets.
TABLE-US-00031 TABLE 26 Percentage of viral reduction caused by
RetroMAD1 micronized powder in simultaneous determined by PCR. Time
RetroMAD1 24 h 48 h RetroMAD1 (in water + NaOH) - 10 .mu.g/ml 98.33
93.06 RetroMAD1 (in water + NaOH) - 15 .mu.g/ml 100.00 100.00
RetroMAD1 (in water) - 5 .mu.g/ml 87.13 85.43
REFERENCES
[0187] 1. Hizer S. E. et. al. (2002). RAPD markers as predictors of
infectious hypodermal and haematopoietic necrosis virus (IHHNV)
resistance in shrimp (Litopenaeus stylirostris). Genome 45(1): 1-7;
[0188] 2. Liu H. et. al. (2009). Antiviral immunity in crustaceans.
Fish Shellfish Immunol. 2009 27(2):79-88. [0189] 3. Argue B. et.
al. (2002). Selective breeding of Pacific white shrimp (Litopenaeus
vannamei) for growth and resistance to Taura Syndrome Virus.
Aquaculture 204(3-4): 447-460. [0190] 4. Aljanabi, S. M. and L.
Martinez, 1997. Universal and rapid salt-extraction of high quality
genomic DNA for PCR-based techniques. Nucleic Acid Res., 25:
4692-4693; [0191] Sambrook and Green, Molecular Cloning: A
Laboratory Manual, Cold Springs Harbor Laboratory (Fourth Edition),
New York (2012), [0192] 5. Tang Y Q, Yuan J, Osapay G et al.
(October 1999). "A cyclic antimicrobial peptide produced in primate
leukocytes by the ligation of two truncated alpha-defensins".
Science 286 (5439): 498-502; [0193] 6. Leonova L, Kokryakov V N,
Aleshina G et al. (September 2001). "Circular minidefensins and
posttranslational generation of molecular diversity". J. Leukoc.
Biol. 70 (3): 461-4. [0194] 7. Wang W et al Activity of alpha- and
theta-defensins against primary isolates of HIV-1. Journal of
Immunology 173(1): 515-520 (2004), [0195] 8. U.S. Pat. No.
8,076,284 B2; [0196] 9. Kim, S. et al, Peptides, 2003, 24, 945-953.
[0197] 10. Fan, J-M., et al, Mol Biotechnol, 2008, 39, 79-86.
[0198] 11. Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA
90: 5873-5877 [0199] 12. Brudno M., Bioinformatics 2003b, 19 Suppl
1:154-162.
Sequence CWU 1
1
431379PRTArtificial SequencePolypeptide sequence of Amatilin 1Met
Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10
15 Ala Gln Pro Ala Met Ala Met Gly Arg Ile Cys Arg Cys Ile Cys Gly
20 25 30 Arg Gly Ile Cys Arg Cys Ile Cys Gly Val Pro Gly Val Gly
Val Pro 35 40 45 Gly Val Gly Gly Ala Thr Gly Ser Asp Val Asn Phe
Asp Leu Ser Thr 50 55 60 Ala Thr Ala Lys Thr Tyr Thr Lys Phe Ile
Glu Asp Phe Arg Ala Thr 65 70 75 80 Leu Pro Phe Ser His Lys Val Tyr
Asp Ile Pro Leu Leu Tyr Ser Thr 85 90 95 Ile Ser Asp Ser Arg Arg
Phe Ile Leu Leu Asp Leu Thr Ser Tyr Ala 100 105 110 Tyr Glu Thr Ile
Ser Val Ala Ile Asp Val Thr Asn Val Tyr Val Val 115 120 125 Ala Tyr
Arg Thr Arg Asp Val Ser Tyr Phe Phe Lys Glu Ser Pro Pro 130 135 140
Glu Ala Tyr Asn Ile Leu Phe Lys Gly Thr Arg Lys Ile Thr Leu Pro 145
150 155 160 Tyr Thr Gly Asn Tyr Glu Asn Leu Gln Thr Ala Ala His Lys
Ile Arg 165 170 175 Glu Asn Ile Asp Leu Gly Leu Pro Ala Leu Ser Ser
Ala Ile Thr Thr 180 185 190 Leu Phe Tyr Tyr Asn Ala Gln Ser Ala Pro
Ser Ala Leu Leu Val Leu 195 200 205 Ile Gln Thr Thr Ala Glu Ala Ala
Arg Phe Lys Tyr Ile Glu Arg His 210 215 220 Val Ala Lys Tyr Val Ala
Thr Asn Phe Lys Pro Asn Leu Ala Ile Ile 225 230 235 240 Ser Leu Glu
Asn Gln Trp Ser Ala Leu Ser Lys Gln Ile Phe Leu Ala 245 250 255 Gln
Asn Gln Gly Gly Lys Phe Arg Asn Pro Val Asp Leu Ile Lys Pro 260 265
270 Thr Gly Glu Arg Phe Gln Val Thr Asn Val Asp Ser Asp Val Val Lys
275 280 285 Gly Asn Ile Lys Leu Leu Leu Asn Ser Arg Ala Ser Thr Ala
Asp Glu 290 295 300 Asn Phe Ile Thr Thr Met Thr Leu Leu Gly Glu Ser
Val Val Glu Phe 305 310 315 320 Pro Trp Ala Leu Trp Lys Thr Met Leu
Lys Glu Leu Gly Thr Met Ala 325 330 335 Leu His Ala Gly Lys Ala Ala
Leu Gly Ala Ala Ala Asp Thr Ile Ser 340 345 350 Gln Gly Thr Gln Val
Pro Gly Val Gly Val Pro Gly Val Gly Lys Leu 355 360 365 Ala Ala Ala
Leu Glu His His His His His His 370 375 21140DNAArtificial
SequenceCoding sequence of Amatilin 2atgaaatacc tgctgccgac
cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60atggccatgg ggcgtatttg
ccgttgcatt tgcggccgtg gcatttgccg ctgcatctgt 120ggcgtgccgg
gtgttggtgt tccgggtgtg ggtggtgcga ccggatccga tgtgaacttt
180gatctgagca ccgcgaccgc gaaaacctat accaaattca tcgaagattt
tcgtgcgacc 240ctgccgttta gccataaagt gtatgatatc ccgctgctgt
atagcaccat tagcgatagc 300cgtcgtttta ttctgctgga tctgaccagc
tatgcgtatg aaaccattag cgtggcgatt 360gatgtgacca acgtgtatgt
ggtggcgtat cgtacccgtg atgtgagcta ctttttcaaa 420gaaagcccgc
cggaagcgta caacattctg tttaaaggca cccgtaaaat taccctgccg
480tataccggca actatgaaaa cctgcagacc gcggcgcata aaattcgtga
aaacatcgat 540ctgggcctgc cggccctgag cagcgcgatt accaccctgt
tttattataa cgcgcagagc 600gcgccgagcg cgctgctggt gctgattcag
accaccgcgg aagcggcgcg ttttaaatat 660attgaacgcc acgtggcgaa
atatgtggcg accaacttta aaccgaacct ggccattatt 720agcctggaaa
accagtggag cgccctgagc aaacaaattt ttctggccca gaaccagggc
780ggcaaatttc gtaatccggt ggatctgatt aaaccgaccg gcgaacgttt
tcaggtgacc 840aatgtggata gcgatgtggt gaaaggcaac attaaactgc
tgctgaacag ccgtgcgagc 900accgcggatg aaaactttat taccaccatg
accctgctgg gcgaaagcgt ggtggaattc 960ccgtgggcgc tgtggaaaac
catgctgaaa gaactgggca ccatggcgct gcatgcgggt 1020aaagcggcgc
tgggtgcggc agcggatacc attagccagg gcacccaggt tccgggcgtg
1080ggcgttccgg gcgttggtaa gcttgcggcc gcactcgagc accaccacca
ccaccactga 114035PRTArtificial SequenceA polypeptide of the present
invention 3Val Pro Xaa Val Gly 1 5 418PRTArtificial SequenceA
polypeptide of the present invention 4Gly Arg Ile Cys Arg Cys Ile
Cys Gly Arg Gly Ile Cys Arg Cys Ile 1 5 10 15 Cys Gly
5268PRTArtificial SequenceA polypeptide of the present invention
5Gly Ser Asp Val Asn Phe Asp Leu Ser Thr Ala Thr Ala Lys Thr Tyr 1
5 10 15 Thr Lys Phe Ile Glu Asp Phe Arg Ala Thr Leu Pro Phe Ser His
Lys 20 25 30 Val Tyr Asp Ile Pro Leu Leu Tyr Ser Thr Ile Ser Asp
Ser Arg Arg 35 40 45 Phe Ile Leu Leu Asp Leu Thr Ser Tyr Ala Tyr
Glu Thr Ile Ser Val 50 55 60 Ala Ile Asp Val Thr Asn Val Tyr Val
Val Ala Tyr Arg Thr Arg Asp 65 70 75 80 Val Ser Tyr Phe Phe Lys Glu
Ser Pro Pro Glu Ala Tyr Asn Ile Leu 85 90 95 Phe Lys Gly Thr Arg
Lys Ile Thr Leu Pro Tyr Thr Gly Asn Tyr Glu 100 105 110 Asn Leu Gln
Thr Ala Ala His Lys Ile Arg Glu Asn Ile Asp Leu Gly 115 120 125 Leu
Pro Ala Leu Ser Ser Ala Ile Thr Thr Leu Phe Tyr Tyr Asn Ala 130 135
140 Gln Ser Ala Pro Ser Ala Leu Leu Val Leu Ile Gln Thr Thr Ala Glu
145 150 155 160 Ala Ala Arg Phe Lys Tyr Ile Glu Arg His Val Ala Lys
Tyr Val Ala 165 170 175 Thr Asn Phe Lys Pro Asn Leu Ala Ile Ile Ser
Leu Glu Asn Gln Trp 180 185 190 Ser Ala Leu Ser Lys Gln Ile Phe Leu
Ala Gln Asn Gln Gly Gly Lys 195 200 205 Phe Arg Asn Pro Val Asp Leu
Ile Lys Pro Thr Gly Glu Arg Phe Gln 210 215 220 Val Thr Asn Val Asp
Ser Asp Val Val Lys Gly Asn Ile Lys Leu Leu 225 230 235 240 Leu Asn
Ser Arg Ala Ser Thr Ala Asp Glu Asn Phe Ile Thr Thr Met 245 250 255
Thr Leu Leu Gly Glu Ser Val Val Glu Phe Pro Trp 260 265
634PRTArtificial SequenceA polypeptide of the present invention
6Ala Leu Trp Lys Thr Met Leu Lys Glu Leu Gly Thr Met Ala Leu His 1
5 10 15 Ala Gly Lys Ala Ala Leu Gly Ala Ala Ala Asp Thr Ile Ser Gln
Gly 20 25 30 Thr Gln 718PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 7Gly Phe Cys Arg Cys Leu Cys Arg Arg Gly Val Cys Arg Cys
Ile Cys 1 5 10 15 Thr Arg 818PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 8Arg Cys Leu Cys Arg Arg Gly Val Cys Arg Cys Leu Cys Arg
Arg Gly 1 5 10 15 Val Cys 918PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 9Arg Cys Ile Cys Thr Arg Gly Phe Cys Arg Cys Ile Cys Thr
Arg Gly 1 5 10 15 Phe Cys 1018PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 10Gly Ile Cys Arg Cys Ile Cys Gly Arg Gly Ile Cys Arg Cys
Ile Cys 1 5 10 15 Gly Arg 1118PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 11Gly Ile Cys Arg Cys Ile Cys Gly Arg Gly Ile Cys Arg Cys
Ile Cys 1 5 10 15 Gly Arg 1218PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 12Arg Ile Cys Arg Cys Ile Cys Gly Arg Arg Ile Cys Arg Cys
Ile Cys 1 5 10 15 Gly Arg 1318PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 13Gly Ile Cys Arg Cys Ile Cys Gly Arg Gly Ile Cys Arg Cys
Ile Cys 1 5 10 15 Gly Arg 1418PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 14Gly Ile Cys Arg Cys Ile Cys Gly Lys Gly Ile Cys Arg Cys
Ile Cys 1 5 10 15 Gly Arg 1518PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 15Gly Ile Cys Arg Cys Tyr Cys Gly Arg Gly Ile Cys Arg Cys
Ile Cys 1 5 10 15 Gly Arg 1618PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 16Gly Ile Cys Arg Cys Ile Cys Gly Arg Gly Ile Cys Arg Cys
Tyr Cys 1 5 10 15 Gly Arg 1718PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 17Gly Tyr Cys Arg Cys Ile Cys Gly Arg Gly Ile Cys Arg Cys
Ile Cys 1 5 10 15 Gly Arg 1818PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 18Gly Ile Cys Arg Cys Ile Cys Gly Arg Gly Tyr Cys Arg Cys
Ile Cys 1 5 10 15 Gly Arg 1918PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 19Gly Ile Cys Tyr Cys Ile Cys Gly Arg Gly Ile Cys Arg Cys
Ile Cys 1 5 10 15 Gly Arg 2018PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 20Gly Ile Cys Ile Cys Ile Cys Gly Tyr Gly Ile Cys Arg Cys
Ile Cys 1 5 10 15 Gly Arg 2118PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 21Gly Ile Cys Ile Cys Ile Cys Gly Arg Gly Ile Cys Tyr Cys
Ile Cys 1 5 10 15 Gly Arg 2218PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 22Gly Ile Cys Ile Cys Ile Cys Gly Arg Gly Ile Cys Tyr Cys
Ile Cys 1 5 10 15 Gly Arg 2318PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 23Arg Gly Cys Ile Cys Arg Cys Ile Gly Arg Gly Cys Ile Cys
Arg Cys 1 5 10 15 Ile Gly 2418PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 24Arg Gly Cys Ile Cys Arg Cys Ile Gly Arg Gly Cys Ile Cys
Arg Cys 1 5 10 15 Ile Gly 2518PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 25Gly Ile Cys Arg Cys Ile Cys Gly Arg Gly Ile Cys Arg Cys
Ile Cys 1 5 10 15 Gly Arg 2618PRTArtificial SequenceA polypeptide
sequence of naturally occurring and synthetic theta Defensin
proteins 26Gly Ile Cys Arg Cys Ile Cys Gly Lys Gly Ile Cys Arg Cys
Tyr Cys 1 5 10 15 Gly Arg 274PRTArtificial SequenceAn example of
fusion proteins according to the present invention 27Gly Gly Gly
Ser 1 28360PRTArtificial SequenceAn example of fusion proteins
according to the present invention 28Ser Phe Gly Leu Cys Arg Leu
Arg Arg Gly Phe Cys Ala His Gly Arg 1 5 10 15 Cys Arg Phe Pro Ser
Ile Pro Ile Gly Arg Cys Ser Arg Phe Val Gln 20 25 30 Cys Cys Arg
Arg Val Trp Val Pro Gly Val Gly Val Pro Gly Val Gly 35 40 45 Gly
Ala Thr Gly Ser Asp Val Asn Phe Asp Leu Ser Thr Ala Thr Ala 50 55
60 Lys Thr Tyr Thr Lys Phe Ile Glu Asp Phe Arg Ala Thr Leu Pro Phe
65 70 75 80 Ser His Lys Val Tyr Asp Ile Pro Leu Leu Tyr Ser Thr Ile
Ser Asp 85 90 95 Ser Arg Arg Phe Ile Leu Leu Asn Leu Thr Ser Tyr
Ala Tyr Glu Thr 100 105 110 Ile Ser Val Ala Ile Asp Val Thr Asn Val
Tyr Val Val Ala Tyr Arg 115 120 125 Thr Arg Asp Val Ser Tyr Phe Phe
Lys Glu Ser Pro Pro Glu Ala Tyr 130 135 140 Asn Ile Leu Phe Lys Gly
Thr Arg Lys Ile Thr Leu Pro Tyr Thr Gly 145 150 155 160 Asn Tyr Glu
Asn Leu Gln Thr Ala Ala His Lys Ile Arg Glu Asn Ile 165 170 175 Asp
Leu Gly Leu Pro Ala Leu Ser Ser Ala Ile Thr Thr Leu Phe Tyr 180 185
190 Tyr Asn Ala Gln Ser Ala Pro Ser Ala Leu Leu Val Leu Ile Gln Thr
195 200 205 Thr Ala Glu Ala Ala Arg Phe Lys Tyr Ile Glu Arg His Val
Ala Lys 210 215 220 Tyr Val Ala Thr Asn Phe Lys Pro Asn Leu Ala Ile
Ile Ser Leu Glu 225 230 235 240 Asn Gln Trp Ser Ala Leu Ser Lys Gln
Ile Phe Leu Ala Gln Asn Gln 245 250 255 Gly Gly Lys Phe Arg Asn Pro
Val Asp Leu Ile Lys Pro Thr Gly Glu 260 265 270 Arg Phe Gln Val Thr
Asn Val Asp Ser Asp Val Val Lys Gly Asn Ile 275 280 285 Lys Leu Leu
Leu Asn Ser Arg Ala Ser Thr Ala Asp Glu Asn Phe Ile 290 295 300 Thr
Thr Met Thr Leu Leu Gly Glu Ser Val Val Asn Ser Cys Ala Ser 305 310
315 320 Arg Cys Lys Gly His Cys Arg Ala Arg Arg Cys Gly Tyr Tyr Val
Ser 325 330 335 Val Leu Tyr Arg Gly Arg Cys Tyr Cys Lys Cys Leu Arg
Cys Val Pro 340 345 350 Gly Val Gly Val Pro Gly Val Gly 355 360
29401PRTArtificial SequenceAn example of fusion proteins according
to the present invention 29Leu Glu Lys Arg Lys Trp Lys Leu Phe Lys
Lys Ile Glu Lys Val Gly 1 5 10 15 Gln Arg Val Arg Asp Ala Val Ile
Ser Ala Gly Pro Ala Val Ala Thr 20 25 30 Val Ala Gln Ala Thr Ala
Leu Ala Lys Asn Val Pro Gly Val Gly Val 35 40 45 Pro Gly Val Gly
Gly Ala Thr Gly Ser Asp Val Ser Phe Arg Leu Ser 50 55 60 Gly Ala
Thr Ser Lys Lys Lys Val Tyr Phe Ile Ser Asn Leu Arg Lys 65 70 75 80
Ala Leu Pro Asn Glu Lys Lys Leu Tyr Asp Ile Pro Leu Val Arg Ser 85
90 95 Ser Ser Gly Ser Lys Ala Thr Ala Tyr Thr Leu Asn Leu Ala Asn
Pro 100 105 110 Ser Ala Ser Gln Tyr Ser Ser Phe Leu Asp Gln Ile Arg
Asn Asn Val 115 120 125 Arg Asp Thr Ser Leu Ile Tyr Gly Gly Thr Asp
Val Ala Val Ile Gly 130 135 140 Ala Pro Ser Thr Thr Asp Lys Phe Leu
Arg Leu Asn Phe Gln Gly Pro 145 150 155 160 Arg Gly Thr Val Ser Leu
Gly Leu Arg Arg Glu Asn Leu Tyr Val Val 165 170 175 Ala Tyr Leu Ala
Met Asp Asn Ala Asn Val Asn Arg Ala Tyr Tyr Phe 180 185 190 Lys Asn
Gln Ile Thr Ser Ala Glu Leu Thr Ala Leu Phe Pro Glu Val 195 200 205
Val Val Ala Asn Gln Lys Gln Leu Glu Tyr Gly Glu Asp Tyr Gln Ala 210
215 220 Ile Glu Lys Asn Ala Lys Ile Thr Thr Gly Asp Gln Ser Arg Lys
Glu 225 230 235 240 Leu Gly Leu Gly Ile Asn Leu Leu Ile Thr Met Ile
Asp Gly Val Asn 245 250 255 Lys Lys Val Arg Val Val Lys Asp Glu Ala
Arg Phe Leu Leu Ile Ala 260 265 270 Ile Gln Met Thr Ala Glu Ala Ala
Arg Phe Arg Tyr Ile Gln Asn Leu 275 280 285 Val Thr Lys Asn Phe Pro
Asn Lys Phe Asp Ser Glu Asn Lys Val Ile 290 295 300 Gln Phe Gln Val
Ser Trp Ser Lys Ile Ser Thr Ala Ile Phe Gly Asp 305 310 315 320 Cys
Lys Asn Gly Val Phe Asn Lys Asp Tyr Asp Phe Gly Phe Gly Lys 325
330 335 Val Arg Gln Ala Lys Asp Leu Gln Met Gly Leu Leu Lys Tyr Leu
Gly 340 345 350 Arg Pro Lys Ser Ser Ser Ile Glu Ala Asn Ser Thr Asp
Asp Thr Ala 355 360 365 Asp Val Leu Val Pro Gly Val Gly Val Pro Gly
Val Gly Lys Thr Cys 370 375 380 Glu Asn Leu Ala Asp Thr Phe Arg Gly
Pro Cys Phe Ala Thr Ser Asn 385 390 395 400 Cys 30297PRTArtificial
SequenceAn example of fusion proteins according to the present
invention 30Met Gly Arg Ile Cys Arg Cys Ile Cys Gly Arg Gly Ile Cys
Arg Cys 1 5 10 15 Ile Cys Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Gly Ser Asp 20 25 30 Val Asn Phe Asp Leu Ser Thr Ala Thr Ala
Lys Thr Tyr Thr Lys Phe 35 40 45 Ile Glu Asp Phe Arg Ala Thr Leu
Pro Phe Ser His Lys Val Tyr Asp 50 55 60 Ile Pro Leu Leu Tyr Ser
Thr Ile Ser Asp Ser Arg Arg Phe Ile Leu 65 70 75 80 Leu Asp Leu Thr
Ser Tyr Ala Tyr Glu Thr Ile Ser Val Ala Ile Asp 85 90 95 Val Thr
Asn Val Tyr Val Val Ala Tyr Arg Thr Arg Asp Val Ser Tyr 100 105 110
Phe Phe Lys Glu Ser Pro Pro Glu Ala Tyr Asn Ile Leu Phe Lys Gly 115
120 125 Thr Arg Lys Ile Thr Leu Pro Tyr Thr Gly Asn Tyr Glu Asn Leu
Gln 130 135 140 Thr Ala Ala His Lys Ile Arg Glu Asn Ile Asp Leu Gly
Leu Pro Ala 145 150 155 160 Leu Ser Ser Ala Ile Thr Thr Leu Phe Tyr
Tyr Asn Ala Gln Ser Ala 165 170 175 Pro Ser Ala Leu Leu Val Leu Ile
Gln Thr Thr Ala Glu Ala Ala Arg 180 185 190 Phe Lys Tyr Ile Glu Arg
His Val Ala Lys Tyr Val Ala Thr Asn Phe 195 200 205 Lys Pro Asn Leu
Ala Ile Ile Ser Leu Glu Asn Gln Trp Ser Ala Leu 210 215 220 Ser Lys
Gln Ile Phe Leu Ala Gln Asn Gln Gly Gly Lys Phe Arg Asn 225 230 235
240 Pro Val Asp Leu Ile Lys Pro Thr Gly Glu Arg Phe Gln Val Thr Asn
245 250 255 Val Asp Ser Asp Val Val Lys Gly Asn Ile Lys Leu Leu Leu
Asn Ser 260 265 270 Arg Ala Ser Thr Ala Asp Glu Asn Phe Ile Thr Thr
Met Thr Leu Leu 275 280 285 Gly Glu Ser Val Val Glu Phe Pro Trp 290
295 31297PRTArtificial SequenceAn example of fusion proteins
according to the present invention 31Met Gly Ser Asp Val Asn Phe
Asp Leu Ser Thr Ala Thr Ala Lys Thr 1 5 10 15 Tyr Thr Lys Phe Ile
Glu Asp Phe Arg Ala Thr Leu Pro Phe Ser His 20 25 30 Lys Val Tyr
Asp Ile Pro Leu Leu Tyr Ser Thr Ile Ser Asp Ser Arg 35 40 45 Arg
Phe Ile Leu Leu Asp Leu Thr Ser Tyr Ala Tyr Glu Thr Ile Ser 50 55
60 Val Ala Ile Asp Val Thr Asn Val Tyr Val Val Ala Tyr Arg Thr Arg
65 70 75 80 Asp Val Ser Tyr Phe Phe Lys Glu Ser Pro Pro Glu Ala Tyr
Asn Ile 85 90 95 Leu Phe Lys Gly Thr Arg Lys Ile Thr Leu Pro Tyr
Thr Gly Asn Tyr 100 105 110 Glu Asn Leu Gln Thr Ala Ala His Lys Ile
Arg Glu Asn Ile Asp Leu 115 120 125 Gly Leu Pro Ala Leu Ser Ser Ala
Ile Thr Thr Leu Phe Tyr Tyr Asn 130 135 140 Ala Gln Ser Ala Pro Ser
Ala Leu Leu Val Leu Ile Gln Thr Thr Ala 145 150 155 160 Glu Ala Ala
Arg Phe Lys Tyr Ile Glu Arg His Val Ala Lys Tyr Val 165 170 175 Ala
Thr Asn Phe Lys Pro Asn Leu Ala Ile Ile Ser Leu Glu Asn Gln 180 185
190 Trp Ser Ala Leu Ser Lys Gln Ile Phe Leu Ala Gln Asn Gln Gly Gly
195 200 205 Lys Phe Arg Asn Pro Val Asp Leu Ile Lys Pro Thr Gly Glu
Arg Phe 210 215 220 Gln Val Thr Asn Val Asp Ser Asp Val Val Lys Gly
Asn Ile Lys Leu 225 230 235 240 Leu Leu Asn Ser Arg Ala Ser Thr Ala
Asp Glu Asn Phe Ile Thr Thr 245 250 255 Met Thr Leu Leu Gly Glu Ser
Val Val Glu Phe Pro Trp Val Pro Gly 260 265 270 Val Gly Val Pro Gly
Val Gly Gly Arg Ile Cys Arg Cys Ile Cys Gly 275 280 285 Arg Gly Ile
Cys Arg Cys Ile Cys Gly 290 295 32313PRTArtificial SequenceAn
example of fusion proteins according to the present invention 32Met
Gly Ser Asp Val Asn Phe Asp Leu Ser Thr Ala Thr Ala Lys Thr 1 5 10
15 Tyr Thr Lys Phe Ile Glu Asp Phe Arg Ala Thr Leu Pro Phe Ser His
20 25 30 Lys Val Tyr Asp Ile Pro Leu Leu Tyr Ser Thr Ile Ser Asp
Ser Arg 35 40 45 Arg Phe Ile Leu Leu Asp Leu Thr Ser Tyr Ala Tyr
Glu Thr Ile Ser 50 55 60 Val Ala Ile Asp Val Thr Asn Val Tyr Val
Val Ala Tyr Arg Thr Arg 65 70 75 80 Asp Val Ser Tyr Phe Phe Lys Glu
Ser Pro Pro Glu Ala Tyr Asn Ile 85 90 95 Leu Phe Lys Gly Thr Arg
Lys Ile Thr Leu Pro Tyr Thr Gly Asn Tyr 100 105 110 Glu Asn Leu Gln
Thr Ala Ala His Lys Ile Arg Glu Asn Ile Asp Leu 115 120 125 Gly Leu
Pro Ala Leu Ser Ser Ala Ile Thr Thr Leu Phe Tyr Tyr Asn 130 135 140
Ala Gln Ser Ala Pro Ser Ala Leu Leu Val Leu Ile Gln Thr Thr Ala 145
150 155 160 Glu Ala Ala Arg Phe Lys Tyr Ile Glu Arg His Val Ala Lys
Tyr Val 165 170 175 Ala Thr Asn Phe Lys Pro Asn Leu Ala Ile Ile Ser
Leu Glu Asn Gln 180 185 190 Trp Ser Ala Leu Ser Lys Gln Ile Phe Leu
Ala Gln Asn Gln Gly Gly 195 200 205 Lys Phe Arg Asn Pro Val Asp Leu
Ile Lys Pro Thr Gly Glu Arg Phe 210 215 220 Gln Val Thr Asn Val Asp
Ser Asp Val Val Lys Gly Asn Ile Lys Leu 225 230 235 240 Leu Leu Asn
Ser Arg Ala Ser Thr Ala Asp Glu Asn Phe Ile Thr Thr 245 250 255 Met
Thr Leu Leu Gly Glu Ser Val Val Glu Phe Pro Trp Val Pro Gly 260 265
270 Val Gly Val Pro Gly Val Gly Ala Leu Trp Lys Thr Met Leu Lys Glu
275 280 285 Leu Gly Thr Met Ala Leu His Ala Gly Lys Ala Ala Leu Gly
Ala Ala 290 295 300 Ala Asp Thr Ile Ser Gln Gly Thr Gln 305 310
33313PRTArtificial SequenceAn example of fusion proteins according
to the present invention 33Met Ala Leu Trp Lys Thr Met Leu Lys Glu
Leu Gly Thr Met Ala Leu 1 5 10 15 His Ala Gly Lys Ala Ala Leu Gly
Ala Ala Ala Asp Thr Ile Ser Gln 20 25 30 Gly Thr Gln Val Pro Gly
Val Gly Val Pro Gly Val Gly Gly Ser Asp 35 40 45 Val Asn Phe Asp
Leu Ser Thr Ala Thr Ala Lys Thr Tyr Thr Lys Phe 50 55 60 Ile Glu
Asp Phe Arg Ala Thr Leu Pro Phe Ser His Lys Val Tyr Asp 65 70 75 80
Ile Pro Leu Leu Tyr Ser Thr Ile Ser Asp Ser Arg Arg Phe Ile Leu 85
90 95 Leu Asp Leu Thr Ser Tyr Ala Tyr Glu Thr Ile Ser Val Ala Ile
Asp 100 105 110 Val Thr Asn Val Tyr Val Val Ala Tyr Arg Thr Arg Asp
Val Ser Tyr 115 120 125 Phe Phe Lys Glu Ser Pro Pro Glu Ala Tyr Asn
Ile Leu Phe Lys Gly 130 135 140 Thr Arg Lys Ile Thr Leu Pro Tyr Thr
Gly Asn Tyr Glu Asn Leu Gln 145 150 155 160 Thr Ala Ala His Lys Ile
Arg Glu Asn Ile Asp Leu Gly Leu Pro Ala 165 170 175 Leu Ser Ser Ala
Ile Thr Thr Leu Phe Tyr Tyr Asn Ala Gln Ser Ala 180 185 190 Pro Ser
Ala Leu Leu Val Leu Ile Gln Thr Thr Ala Glu Ala Ala Arg 195 200 205
Phe Lys Tyr Ile Glu Arg His Val Ala Lys Tyr Val Ala Thr Asn Phe 210
215 220 Lys Pro Asn Leu Ala Ile Ile Ser Leu Glu Asn Gln Trp Ser Ala
Leu 225 230 235 240 Ser Lys Gln Ile Phe Leu Ala Gln Asn Gln Gly Gly
Lys Phe Arg Asn 245 250 255 Pro Val Asp Leu Ile Lys Pro Thr Gly Glu
Arg Phe Gln Val Thr Asn 260 265 270 Val Asp Ser Asp Val Val Lys Gly
Asn Ile Lys Leu Leu Leu Asn Ser 275 280 285 Arg Ala Ser Thr Ala Asp
Glu Asn Phe Ile Thr Thr Met Thr Leu Leu 290 295 300 Gly Glu Ser Val
Val Glu Phe Pro Trp 305 310 34328PRTArtificial SequenceAn example
of fusion proteins according to the present invention 34Val Pro Gly
Val Gly Val Pro Gly Val Gly Lys Trp Cys Phe Arg Val 1 5 10 15 Cys
Tyr Arg Gly Ile Cys Tyr Arg Arg Cys Arg Val Pro Gly Val Gly 20 25
30 Val Pro Gly Val Gly Gly Ala Thr Gly Ser Asp Val Asn Phe Asp Leu
35 40 45 Ser Thr Ala Thr Ala Lys Thr Tyr Thr Lys Phe Ile Glu Asp
Phe Arg 50 55 60 Ala Thr Leu Pro Phe Ser His Lys Val Tyr Asp Ile
Pro Leu Leu Tyr 65 70 75 80 Ser Thr Ile Ser Asp Ser Arg Arg Phe Ile
Leu Leu Asn Leu Thr Ser 85 90 95 Tyr Ala Tyr Glu Thr Ile Ser Val
Ala Ile Asp Val Thr Asn Val Tyr 100 105 110 Val Val Ala Tyr Arg Thr
Arg Asp Val Ser Tyr Phe Phe Lys Glu Ser 115 120 125 Pro Pro Glu Ala
Tyr Asn Ile Leu Phe Lys Gly Thr Arg Lys Ile Thr 130 135 140 Leu Pro
Tyr Thr Gly Asn Tyr Glu Asn Leu Gln Thr Ala Ala His Lys 145 150 155
160 Ile Arg Glu Asn Ile Asp Leu Gly Leu Pro Ala Leu Ser Ser Ala Ile
165 170 175 Thr Thr Leu Phe Tyr Tyr Asn Ala Gln Ser Ala Pro Ser Ala
Leu Leu 180 185 190 Val Leu Ile Gln Thr Thr Ala Glu Ala Ala Arg Phe
Lys Tyr Ile Glu 195 200 205 Arg His Val Ala Lys Tyr Val Ala Thr Asn
Phe Lys Pro Asn Leu Ala 210 215 220 Ile Ile Ser Leu Glu Asn Gln Trp
Ser Ala Leu Ser Lys Gln Ile Phe 225 230 235 240 Leu Ala Gln Asn Gln
Gly Gly Lys Phe Arg Asn Pro Val Asp Leu Ile 245 250 255 Lys Pro Thr
Gly Glu Arg Phe Gln Val Thr Asn Val Asp Ser Asp Val 260 265 270 Val
Lys Gly Asn Ile Lys Leu Leu Leu Asn Ser Arg Ala Ser Thr Ala 275 280
285 Asp Glu Asn Phe Ile Thr Thr Met Thr Leu Leu Gly Glu Ser Val Val
290 295 300 Asn Val Pro Gly Val Gly Val Pro Gly Val Gly His Gly Val
Ser Gly 305 310 315 320 His Gly Gln His Gly Val His Gly 325
35336PRTArtificial SequenceAn example of fusion proteins according
to the present invention 35Val Pro Gly Val Gly Val Pro Gly Val Gly
Phe Leu Pro Leu Leu Ala 1 5 10 15 Gly Leu Ala Ala Asn Phe Leu Pro
Thr Ile Ile Cys Phe Ile Ser Tyr 20 25 30 Lys Cys Val Pro Gly Val
Gly Val Pro Gly Val Gly Gly Ala Thr Gly 35 40 45 Ser Asp Val Asn
Phe Asp Leu Ser Thr Ala Thr Ala Lys Thr Tyr Thr 50 55 60 Lys Phe
Ile Glu Asp Phe Arg Ala Thr Leu Pro Phe Ser His Lys Val 65 70 75 80
Tyr Asp Ile Pro Leu Leu Tyr Ser Thr Ile Ser Asp Ser Arg Arg Phe 85
90 95 Ile Leu Leu Asn Leu Thr Ser Tyr Ala Tyr Glu Thr Ile Ser Val
Ala 100 105 110 Ile Asp Val Thr Asn Val Tyr Val Val Ala Tyr Arg Thr
Arg Asp Val 115 120 125 Ser Tyr Phe Phe Lys Glu Ser Pro Pro Glu Ala
Tyr Asn Ile Leu Phe 130 135 140 Lys Gly Thr Arg Lys Ile Thr Leu Pro
Tyr Thr Gly Asn Tyr Glu Asn 145 150 155 160 Leu Gln Thr Ala Ala His
Lys Ile Arg Glu Asn Ile Asp Leu Gly Leu 165 170 175 Pro Ala Leu Ser
Ser Ala Ile Thr Thr Leu Phe Tyr Tyr Asn Ala Gln 180 185 190 Ser Ala
Pro Ser Ala Leu Leu Val Leu Ile Gln Thr Thr Ala Glu Ala 195 200 205
Ala Arg Phe Lys Tyr Ile Glu Arg His Val Ala Lys Tyr Val Ala Thr 210
215 220 Asn Phe Lys Pro Asn Leu Ala Ile Ile Ser Leu Glu Asn Gln Trp
Ser 225 230 235 240 Ala Leu Ser Lys Gln Ile Phe Leu Ala Gln Asn Gln
Gly Gly Lys Phe 245 250 255 Arg Asn Pro Val Asp Leu Ile Lys Pro Thr
Gly Glu Arg Phe Gln Val 260 265 270 Thr Asn Val Asp Ser Asp Val Val
Lys Gly Asn Ile Lys Leu Leu Leu 275 280 285 Asn Ser Arg Ala Ser Thr
Ala Asp Glu Asn Phe Ile Thr Thr Met Thr 290 295 300 Leu Leu Gly Glu
Ser Val Val Asn Val Pro Gly Val Gly Val Pro Gly 305 310 315 320 Val
Gly Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys 325 330
335 36325PRTArtificial SequencePolypeptide sequence of RetroGAD1
36Gly Ile Cys Arg Cys Ile Gly Arg Gly Ile Cys Arg Cys Ile Cys Gly 1
5 10 15 Arg Val Pro Gly Val Gly Val Pro Gly Val Gly Gly Ala Thr Gly
Ser 20 25 30 Gly Leu Asp Thr Val Ser Phe Ser Thr Lys Gly Ala Thr
Tyr Ile Thr 35 40 45 Tyr Val Asn Phe Leu Asn Glu Leu Arg Val Lys
Leu Lys Pro Glu Gly 50 55 60 Asn Ser His Gly Ile Pro Leu Leu Arg
Lys Lys Cys Asp Asp Pro Gly 65 70 75 80 Lys Cys Phe Val Leu Val Ala
Leu Ser Asn Asp Asn Gly Gln Leu Ala 85 90 95 Glu Ile Ala Ile Asp
Val Thr Ser Val Tyr Val Val Gly Tyr Gln Val 100 105 110 Arg Asn Arg
Ser Tyr Phe Phe Lys Asp Ala Pro Asp Ala Ala Tyr Glu 115 120 125 Gly
Leu Phe Lys Asn Thr Ile Lys Thr Arg Leu His Phe Gly Gly Ser 130 135
140 Tyr Pro Ser Leu Glu Gly Glu Lys Ala Tyr Arg Glu Thr Thr Asp Leu
145 150 155 160 Gly Ile Glu Pro Leu Arg Ile Gly Ile Lys Lys Leu Asp
Glu Asn Ala 165 170 175 Ile Asp Asn Tyr Lys Pro Thr Glu Ile Ala Ser
Ser Leu Leu Val Val 180 185 190 Ile Gln Met Val Ser Glu Ala Ala Arg
Phe Thr Phe Ile Glu Asn Gln 195 200 205 Ile Arg Asn Asn Phe Gln Gln
Arg Ile Arg Pro Ala Asn Asn Thr Ile 210 215 220 Ser Leu Glu Asn Lys
Trp Gly Lys Leu Ser Phe Gln Ile Arg Thr Ser 225 230 235 240 Gly Ala
Asn Gly Met Phe Ser Glu Ala Val Glu Leu Glu Arg Ala Asn 245 250 255
Gly Lys Lys Tyr Tyr Val Thr Ala Val Asp Gln Val Lys Pro Lys Ile
260
265 270 Ala Leu Leu Lys Phe Val Asp Lys Asp Pro Lys Gly Leu Trp Ser
Lys 275 280 285 Ile Lys Glu Ala Ala Lys Ala Ala Gly Lys Ala Ala Leu
Asn Ala Val 290 295 300 Thr Gly Leu Val Asn Gln Gly Asp Gln Pro Ser
Val Pro Gly Val Gly 305 310 315 320 Val Pro Gly Val Gly 325
371185DNAArtificial SequenceCoding sequence of Amatilin
37gggcagtgag cggaaggccc atgaggccag ttaattaaga ggtaccgaat tctcattcgg
60tttgtgtaga ttgagaagag gtttctgtgc tcacggtaga tgtagattcc catccatccc
120aatcggtaga tgttccagat tcgttcagtg ttgtagaaga gtttgggtcc
caggtgttgg 180tgttccaggt gttggaggtg ctactggttc tgatgttaac
ttcgacttgt ccactgctac 240tgctaagact tacactaagt tcatcgagga
cttcagagct actttgccat tctcccacaa 300ggtttacgac atccctttgt
tgtactccac tatctccgac tccagaagat tcatcttgtt 360gaacttgact
tcctacgctt acgagactat ctccgttgct atcgacgtta caaacgttta
420cgttgttgct tacagaacta gagatgtttc ctacttcttc aaagagtccc
caccagaggc 480ttacaacatc ttgttcaagg gtactagaaa gatcactttg
ccatacactg gtaactacga 540gaacttgcag actgctgctc acaagatcag
agagaacatc gacttgggtt tgccagcttt 600gtcctccgct atcactactt
tgttctacta caacgctcag tccgctccat ccgctttgtt 660ggttttgatc
cagactactg ctgaggctgc tagattcaag tacatcgaga gacacgttgc
720taagtacgtt gctacaaact tcaagccaaa cttggctatc atctccttgg
agaaccagtg 780gtctgctttg tccaagcaga tcttcttggc tcaaaaccag
ggtggtaagt tcagaaaccc 840agtcgacttg atcaagccaa ccggtgagag
attccaggtt actaatgttg actccgacgt 900tgttaagggt aacatcaagt
tgttgttgaa ctccagagct tccactgctg acgagaactt 960catcactact
atgactttgt tgggtgagtc cgttgttaac tcctgtgctt ccagatgtaa
1020gggtcactgt agagctagaa gatgtggtta ctacgtttcc gttctgtaca
gaggtagatg 1080ttactgtaaa tgtttgagat gtgtccccgg tgttggagtc
cctggtgtcg gtgcggccgc 1140gagctcatgg cgcgcctagg ccttgacggc
cttccgccaa ttcgc 1185381084DNAArtificial SequenceCoding sequence
for RetroGAD1 38cgaattggcg gaaggccgtc aaggccacgt gtcttgtcca
ggtaccgaat tcggaatctg 60tagatgcatc tgcggtagag gtatctgcag atgtatttgt
ggaagagtcc caggtgttgg 120tgttccaggt gttggaggtg ctactggttc
tggtttggac actgtttcat tctccactaa 180gggtgctact tacatcactt
acgttaactt tttgaacgag ttgagagtta agttgaagcc 240agagggtaac
tcccacggta tccctttgtt gagaaagaag tgtgacgacc caggtaagtg
300tttcgttttg gttgctttgt ccaacgacaa cggtcagttg gctgagattg
ctatcgacgt 360tacttccgtt tacgttgttg gttaccaggt tagaaacaga
tcctacttct tcaaggacgc 420tccagacgct gcttacgaag gtttgttcaa
gaacactatc aagactagat tgcacttcgg 480tggttcctac ccatctttgg
aaggtgagaa ggcttacaga gagactactg acttgggtat 540cgagccattg
agaatcggta tcaagaagtt ggacgagaac gctatcgaca actacaagcc
600aactgagatc gcttcctcct tgttggttgt tatccagatg gtttccgagg
ctgctagatt 660cactttcatc gagaaccaga tcagaaacaa cttccagcag
agaatcagac cagctaacaa 720cactatttcc ttggagaaca agtggggtaa
gttgtccttc cagatcagaa catccggtgc 780taacggtatg ttctctgagg
ctgttgagtt ggagagagct aacggtaaga agtactacgt 840tactgctgtt
gaccaggtta agccaaagat cgctttgttg aagttcgttg acaaggaccc
900aaagggtttg tggtccaaga tcaaagaggc tgctaaggct gctggtaagg
ctgctttgaa 960tgctgttact ggtttggtta accagggtga ccaaccatct
gtccctggtg ttggagtccc 1020tggtgtcggt gcggccgcga gctctggagc
acaagactgg cctcatgggc cttccgctca 1080ctgc 108439998DNAArtificial
SequenceCoding sequence of Tampal 1 39ggatccgttc cgggtgtggg
tgttccgggt gttggtaaat ggtgtttcgt gtttgttatc 60gcggtatttg ttatcgtcgt
tgtcgtgtgc caggcgttgg cgttccaggc gtgggtggtg 120caaccggtag
tgatgttaat tttgatctga gcaccgcaac cgcaaaaacc tataccaaat
180ttatcgaaga ttttcgtgca accctgccgt ttagccataa agtttatgat
attccgctgc 240tgtatagcac cattagcgat agccgtcgtt ttattctgct
gaatctgacc agctatgcct 300atgaaaccat tagcgttgca attgatgtga
ccaatgttta tgttgttgca tatcgtaccc 360gtgatgtgag ctattttttc
aaagaaagcc ctccggaagc ctataacatt ctgtttaaag 420gcacccgcaa
aatcaccctg ccgtataccg gtaattatga aaatctgcag accgcagcac
480ataaaattcg cgaaaatatt gatctgggtc tgcctgcact gagcagcgca
attaccaccc 540tgttttatta caatgcacag agcgcaccga gcgcactgct
ggttctgatt cagaccaccg 600cagaagcagc acgctttaaa tacattgaac
gtcatgttgc caaatacgtg gccaccaact 660ttaaaccgaa tctggcaatt
attagcctgg aaaatcagtg gtcagcactg agcaaacaaa 720tttttctggc
acagaatcag ggtggcaaat ttcgtaatcc ggttgatctg attaaaccga
780ccggtgaacg ttttcaggtt accaatgttg atagtgatgt ggtgaaaggc
aacattaaac 840tgctgctgaa tagccgtgca agcaccgcag atgaaaactt
tattaccacc atgaccctgc 900tgggtgaaag cgttgttaat gttcctggtg
ttggcgtgcc tggtgttggt catggtgtta 960gcggtcatgg tcagcatggt
gttcatggtt aaaagctt 9984020DNAArtificial SequenceSense Primer F1 of
Example 12 40atggatttgg caacctaaca 204119DNAArtificial
SequenceSense primer F2 of Example 12 41aattcgtgga gagaggtcc
194220DNAArtificial SequenceSense primer F3 of Example 12
42atctctaccg tcacacagcc 204323DNAArtificial SequenceAntisense
primer R1 of Example 12 43gaagatttta atgtccttgc tcg 23
* * * * *
References